DNA sequence-based HLA typing method

ABSTRACT

The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class II and Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of DRB, DQB, DQA, DPB, DPA, HLA-A, HLA-B and HLA-C- transcripts enzymatically amplified using a limited number of non-allele-specific oligonucleotides. Total cellular RNA from peripheral blood mononuclear cells is reverse transcribed using antisense primers, specific for different locus (DQB, DQA, DPA or DPB) or group of loci (DRB1-5, or HLA-A and HLA-B and HLA-C). The synthesized cDNA molecules are then enzymatically amplified using different combinations of oligonucleotides for each locus and directly sequenced with Taq polymerase using an internal oligonucleotide. The sequenced genes are then analyzed.

GOVERNMENT SUPPORT

This invention was made with Government support under grant number DK36828 by the U.S. National Institutes of Health. The Government hascertain rights in the invention.

This is a continuation of application Ser. No. 07/833,668, nowabandoned, filed Feb. 18, 1992, which is a continuation-in-part ofapplication Ser. No. 07/665,960, filed on Mar. 6, 1991.

TECHNICAL FIELD

The present invention relates to a process for determining genotypes ofhighly polymorphic systems, such as the major histocompatibility complexof humans, including Class I and Class II HLA genes. Specifically, themethod of the present invention involves amplifying the alleles carriedby any given individual at a gene locus or loci of interest bypolymerase chain reaction with conserved and non-conservedoligonucleotide primers. The polymerase chain reaction products aredirectly sequenced followed by evaluation of the resulting nucleic acidladders to determine the genotype of sample nucleic acid.

BACKGROUND OF THE INVENTION

The major histocompatibility complex (MHC) includes the human leukocyteantigens (HLA) gene complex which is located on the short arm of humanchromosome six. This region encodes cell-surface proteins which regulatethe cell-cell interactions of the immune response. The various HLA ClassI loci encode the HLA antigens, 44,000 dalton polypeptides whichassociate with B-2 microglobulin at the cell surface. The Class Imolecules are involved in the recognition of target cells by cytotoxic Tlymphocytes. HLA Class II loci encode cell surface heterodimers,composed of proteins of 29,000 and 34,000 daltons, respectively. TheseClass II molecules are also involved in the recognition of target cellsby helper T lymphocytes.

The HLA-A, HLA-B, and HLA-C loci of the HLA Class I region as well asthe HLA-DRB, HLA-DQB, HLA-DQA, HLA-DPB and HLA-DPA loci of the HLA ClassII region exhibit an extremely high degree of polymorphism. The WHOnomenclature committee for factors of the HLA system [Marsh and Bodmer,Immunogenetics, 31:131 (1990)] designated 25 alleles of HLA-A(HLA-A-0101, A-0201, etc.), 32 alleles of HLA-B, and 11 alleles ofHLA-C, 43 HLA-DRB alleles, 13 HLA-DQB alleles, 8 HLA-DQA alleles, 4HLA-DPA alleles and 19 HLA-DPB alleles. Since this high degree ofpolymorphism is thought to relate to the function of the HLA molecules,much effort has gone into determining its molecular basis and thefunctional implications of its polymorphisms (i.e., in transplantation).With the cloning of certain HLA genes this effort has extended to theDNA level.

The Class II genes of the HLA-D region on the short arm of humanchromosome six constitute one of the most polymorphic genetic systemsknown [Bach, Immunol. Today, 6:89 (1985)]. The HLA Class II molecules(DR, DQ and DP) are heterodimeric glycoproteins composed of twonon-covalently associated chains (alpha and beta) which serve asrestricting elements in nominal antigen presentation in the context ofself [Zinkernagel and Doherty, Nature, 248:701 (1974)] or as foreignantigens in alloresponses [Bach and Van Rood, N. Engl. J. Med., 295:806(1976)].

Allelic polymorphism of the HLA-D region encoded specificities can bedetermined by serological methods for phenotyping, mixed lymphocytecultures using homozygous typing cells, primed lymphocyte testing,determination of restriction fragment length polymorphisms and, morerecently, oligotyping [Bach, supra (1985); Bidwell, Immunol. Today, 9:18(1988); Tiercy et al., Proc. Natl. Acad. Sci. USA, 85:198 (1988)].Present efforts focus largely on the development of molecular approachesto typing, such as RFLP and oligotyping [Bidwell, supra (1988); Tiercyet al., supra (1988); Erlich and Bugawan, in PCR Techniques, H. A.Erlich, ed., Stockton Press, New York (1989)].

The cloning and sequencing of several HLA-DR, DQ, and -DP alleles hasrevealed that their amino acid polymorphisms are located inhypervariable regions of their N-terminal domains, encoded by the secondexon of DRB1, DRB3/4/5, DQA1 and DQB1, DPA1 and DPB1 genes [Marsh andBodmer, supra (1990); Todd et al., Nature, 329:599 (1987)]. Thisinformation has allowed the design of allele-specific oligonucleotideswhich can be used in the characterization of the known HLA Class IIpolymorphisms by means of their hybridization to DNA on a solid support(oligomer typing) or for sequencing [Tiercy et al., supra (1988); Erlichand Bugawan, supra, (1989); Todd et al., supra (1987); Saiki et al.,Science, 230:1350 (1985); Mullis and Faloona, Methods Enzymol., 155:335(1987); Saiki et al., Nature, 324:163 (1986); Scharf et al., Science,233:1076 (1986); Gyllenstein and Erlich, Proc. Natl. Acad. Sci. USA,85:7652 (1988)]. Oligonucleotide typing, although rapid, requires theuse of a rather large number of oligonucleotides for each locus andcannot detect previously unidentified sequence polymorphisms, likely toexist in non-Caucasian populations; further, the approach may not beeasily applicable to and may not be practical for the analysis of ClassI polymorphisms. Direct sequencing of single-stranded DNA generated byPCR using allele-specific oligonucleotides has been successfully used toexamine polymorphism at DQA1 locus [Gyllenstein and Erlich, supra(1988)]. Application of this approach to DRB genes is, however,problematic due to the strong sequence homology among DRB1, DRB3, DRB4and DRB5 genes and the presence of up to four different versions of eachof these genes in most individuals (isotypic complexity). The verycomplex ladders generated by direct sequencing make this present processimpractical for accurate and rapid determination of HLA types. Thus,direct sequencing of HLA-PCR products has been limited to previousknowledge of the HLA types carried by a given individual and as such isnot suitable for routine HLA typing [Bach, supra (1985); Bidwell, supra(1988); Tiercy et al., supra (1988); Erlich and Bugawan, supra (1989)].

Currently, HLA typing is routinely done in connection with many medicalprocedures, e.g., organ transplantation. Rejection of organ grafts isbelieved to be diminished if the HLA alleles of donor and recipient areidentical. The numerous alleles of HLA genes in the population also makeHLA typing useful for paternity testing. However, the currentlyavailable techniques are incapable of differentiating among all of thepolymorphisms associated with the alleles at Class I and Class II HLAloci. Other drawbacks to current HLA typing are the availability ofstandard sera necessary to conduct serological tests, the speed ofobtaining test results (i.e., MLC takes 5-7 days), and that only thealready known HLA types, but not new polymorphisms, are detected bythese techniques. In the case of tissue typing in organ transplants andin relatively high volume genetic evaluations, such as paternitytesting, the length of time associated with current HLA typingtechniques causes unnecessary delay and the results may not be highlyaccurate.

Accordingly, there is a need for a method to determine genomicinformation in highly polymorphic systems, such as the HLA gene complex,that addresses the limitations imposed by previous methods. That is, inthe case of the HLA gene complex, a system that is capable ofdetermining the nucleotide sequences of the genes carried by any givenindividual without the need to have previous knowledge of his or her HLAtypes as defined by other methods. Furthermore, the invention avoids theuse of oligonucleotides specific for each known allele. The technique wepresent is rapid, requires the use of only a small number ofoligonucleotide primers, and can readily detect new sequence variantsunidentifiable with more conventional approaches. This system isexemplified by its applicability to the analysis of Class II as well asClass I and Class III genes and is automatable.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining the nucleicacid sequence of one or more polymorphic genes of a subject byamplifying and direct sequencing genomic or complementary DNA moleculesfor each allele at each gene locus to be sequenced using conserved andnon-conserved (non-allele-specific) oligonucleotide primers. In a broadsense, the method of the present invention involves sequence-basedtyping (SBT) which provides for unequivocal determination of geneticpolymorphism at any genetic locus of interest by direct, simultaneous,sequence analysis of both genomic DNA or expressed (RNA) copies of sucha locus. SBT can be employed to determine genetic polymorphism at one ormore genetic loci of interest, regardless of the complexity of thepolymorphism at these loci, including, for example: (1) simplehomozygosity or heterozygosity of a unique locus, as exemplified by DQAor the like; (2) isotypic complexity due to multiple, closely relatedand closely linked copies of a locus, as exemplified by DRB or the like;and (3) intra-allelic complexity at a locus compounded by interlocuscomplexity, such as Class I genes or the like. Most known human geneticpolymorphisms are of the first, and simplest, type.

Use of the SBT method provides overlapping sequence data comprised ofonly the copies of the locus of interest as is exemplified by each ofthe types of HLA loci. The SBT strategy is designed to ensure selectionof a given locus with equal representation of each copy of that locus byequal amplification and direct sequencing of mixtures of both alleles ofthat locus and direct interpretation of the overlapping sequencingpatterns generated by this approach. Thus, providing a method fordetermining genetic polymorphism at one or more genetic loci of interestwhich can be employed, for example, in HLA typing, detection,evaluation, and/or characterization of genetic diseases such as, forexample, sickle cell anemia, cystic fibrosis, Thalassemia, and the like,and detection, evaluation, and/or characterization of polymorphism ingenetic loci associated with various cancers such as p53, Ras, myc,associated with carcinomas, leukemias, sarcomas or the like.

Use of the method according to the present invention is exemplified by asystem providing for rapid and accurate determination of a majorhistocompatibility complex class genotype of a subject in a sample(e.g., Class I or Class II). Most particularly, the method is directedto determining at least one HLA Class II gene locus including DRB1,DRB3, DRB4, DRB5, DQB1, DQA1, DPA1 and DPB1 genes. In the case of ClassI genotypes, the method is envisioned as being useful to determine A, B,and C loci genes.

To determine a gene locus nucleic acid sequence polymorphism with themethod of the present invention, nucleic acid (RNA or DNA) from a sampleis isolated. In the case of RNA, cDNA molecules for each allele of atleast one gene locus to be sequenced are synthesized by employing alocus-specific oligonucleotide primer that anneals to a conserved regionof each allele of each gene locus. According to the present invention,the sample nucleic acid sequence is determined by: amplifying the cDNAmolecules or genomic DNA by polymerase chain reaction to generatesufficient product for each allele of each gene locus to be sequenced,with all of the alleles for each gene locus and chromosome to besequenced being amplified with at least one conserved oligonucleotideprimer pair, and at least one of the alleles of each gene locus andchromosome to be sequenced being amplified with at least onenon-conserved oligonucleotide primer and at least one conserved primer;preparing the products of each PCR for sequencing (clean); sequencingdirectly the products of each polymerase chain reaction product todetect each allele at each gene locus of each chromosome, with an enzymeappropriate for DNA sequencing, such as Taq polymerase and a conservedprimer specific for each locus that is sequenced; and analyzing eachsequenced product for each locus and primer combination(s) to determinethe genotype of the subject.

In a preferred embodiment of the present invention the sequence of eachpolymerase chain reaction product for each allele of each gene locus isdetermined by analyzing each nucleic acid single and/or overlappingladder generated for each directly sequenced polymerase chain reactionproduct. The analysis is conducted by comparing the nucleotide sequenceof each allele of each gene locus sequence to known sequences for eachlocus, followed by comparing the sequence of each gene locus amplifiedwith the non-conserved/conserved oligonucleotide primer pair to thenucleotide sequence of each allele of the gene locus amplified with aconserved oligonucleotide primer pair. Comparison of nucleic acidladders for sequenced alleles can be conducted visually or usingcomputer software.

In a preferred embodiment, the process of the invention is automated foruse in rapid genotype determinations, including diagnosis of geneticdiseases. Automation of the process includes isolating the samplenucleic acid with an RNA/DNA extractor; amplifying the synthesized cDNAmolecule or the isolated DNA molecule by polymerase chain reaction usinga thermocycler to generate the polymerase chain reaction products;sequencing the polymerase chain reaction products in an automatedsequencing apparatus; and analyzing each sequenced polymerase chainreaction product with the computer having a database with allelicsequence information and the capacity to conduct the appropriatesubstraction algorithm for comparing the polymerase chain reactionproduct sequence for each allele amplified with a conservedoligonucleotide primer pair to the nucleic acid sequence of each allelesequenced with a non-conserved/conserved oligonucleotide primer pair.

The invention further relates to specific groups of oligonucleotideprimers useful in the steps of cDNA synthesis, cDNA/genomic DNAamplification by polymerase chain reaction and direct sequencing of thepolymerase chain reaction products to determine the nucleotide sequenceof each of the alleles at each locus of each chromosome that isamplified. Useful single strand DNA oligonucleotide primers aredescribed in Table 1 herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of the cDNA/PCR/Sequencing experiments for DRB(DRB1, DRB3, DRB4 and DRB5), DQA1, DQB1, DPA1 and DPB1 genes.

FIG. 1B shows a schematic of the primer binding sites on a DRBtranscript. Stippled boxes represent primers used in the cDNA synthesisreactions; black boxes represent conserved (or Type 1) primers, used forPRR; checked boxes represent non-conserved (or Type 2) primers, al/soused for PCR; and blank boxes represent sequencing primers.

FIG. 1C shows a schematic of the primer binding sites on a DQBtranscript. Stippled boxes represent primers used in the cDNA synthesisreactions; black boxes represent conserved (or Type 1) primers, used forPRR; checked boxes represent non-conserved (or Type 2) primers, alsoused for PCR; and blank boxes represent sequencing primers.

FIG. 1D shows a schematic of the primer binding sites on a DQAtranscript. Stippled boxes represent primers used in the cDNA synthesisreactions; black boxes represent conserved (or Type 1) primers, used forPRR; and blank boxes represent sequencing primers.

FIG. 1E shows a schematic of the primer binding sites on a DPBtranscript. Stippled boxes represent primers used in the cDNA synthesisreactions; black boxes represent conserved (or Type 1) primers, used forPRR; and blank boxes represent sequencing primers.

FIG. 1F shows a schematic of the primer binding sites on a DPAtranscript. Stippled boxes represent primers used in the cDNA synthesisreactions; black boxes represent conserved (or Type 1) primers, used forPRR; and blank boxes represent sequencing primers.

FIG. 1G shows a schematic of the primer binding sites on DQB1, DRB, DPB1and DPA1 genes in their germline configuration. Only those primersexclusively used for genomic DNA samples are shown in the Figure.

FIG. 2A shows a flow-chart of the procedure for peripheral bloodsamples. Each reaction is performed in a different test tube. Thereactions are named with capital letter in parenthesis; these letterscorrespond to those shown in Table II (combinations ofprimers/reaction). Only the "routine" combinations of primers are shownin this Figure.

FIG. 2B is a flow-chart of the procedure for forensic samples, where DNAis usually the only available genetic material to work with. DNA inthese situations is usually isolated from hair, sperm, blood stains,etc. The combinations of primers per reaction shown in the Figurecorrespond to the "routine" combinations only.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "gene" refers to a segment of DNA, composed ofa transcribed region and a regulatory sequence that makes possible atranscription. The term "gene locus" refers to the specific place on thechromosome where a gene is located. The term "allele" refers to themultiple forms of a gene that can exist at a single gene locus at asingle chromosome and are distinguishable from the other possiblealleles by their differing effects on phenotype (detectable outwardmanifestations of a specific genotype). "Haplotype" refers to thespecific allele composition of the genes at multiple loci on the samechromosome. As used herein the term "genotype" refers to the specificallelic composition of a gene at multiple linked loci at each chromosome(2 haplotypes).

The term "oligonucleotide" as used herein refers to a molecule havingtwo or more deoxyribonucleotides or ribonucleotides, preferably morethan three deoxyribonucleotides. The exact number of nucleotides in themolecule will depend on the function of the specific oligonucleotidemolecule. As used herein the term "primer" refers to a single strandedDNA oligonucleotide sequence, preferably produced synthetically which iscapable of acting as a point of initiation for synthesis of a primerextension product which is complementary to a nucleic acid strand to becopied or a point of initiation for sequencing a DNA molecule. In thecase of primers intended for use in synthesizing cDNA or amplifying cDNAor genomic DNA molecules by polymerase chain reaction products, thelength and sequence of the primer must be sufficient to prime thesynthesis of extension products in the presence of a polymerizationenzyme. Preferably, the length of the primer is from about 5-50nucleotides, more preferably from about 5-20 nucleotides. Specificlength and sequence of the primer will depend on complexity of requiredDNA or RNA target templates, as well as conditions of primer employmentsuch as temperature, ionic strength, and MgCl₂ concentration. Whennested primers are used for sequencing, the number of base pairsseparating the amplification and sequencing primers on the DNA templateare also important considerations.

As used herein, "conserved oligonucleotide primer" (Type 1) refers to anoligonucleotide molecule that corresponds to a region of high DNAsequence conservation (i.e. less than 1-2 nucleotide variations). Whilethe conserved primer need not correspond exactly to the nucleotidetemplate to which it anneals, the conserved primer will have minimal,preferably less than one mismatch with the target nucleotide template.Functionally, conserved primers are capable of equally priming thetarget nucleotide (cDNA, PCR product, etc.) at high stringencyconditions. In contrast to this, as used herein, "non-conservedoligonucleotide primer" (Type 2) refers to an oligonucleotide moleculethat has an intended number of mismatches with the possible targetnucleotide sequences. The intended number of mismatches can vary with apreferred number of mismatches being about 1-12. Non-conserved primersare characterized by their selective binding to a limited number ofalleles at a given locus or at a group of highly homologous loci. Thenon-conserved primer will bind to the more complementary allele or groupof alleles (two or less than two) (i.e., fewer number of mismatchesbetween primer and target template sequence). The specific combinationsof conserved and non-conserved primers and the number of reactions perlocus or loci used herein are specifically designed to obtain highlyaccurate results with minimal expenditure of time and cost.

The present invention is directed to a process for determining thesequences of the alleles of polymorphic gene systems carried by anygiven individual, such as, for example, the human HLA system, genesrelated to different human genetic disorders, such as sickle cellanemia, cystic fibrosis, or the like, as well as gene systems associatedwith various cancers, such as p53, myc, or the like. The presentinvention is exemplified by its utility for determining polymorphism atHLA loci, particularly Class II and Class I genes, the most polymorphichuman genetic loci known today, using enzymatic amplification and directsequencing of the gene cDNA molecules using a limited number of primersand avoiding the use of allele specific oligonucleotides as much aspossible. The present method is particularly well suited to determiningallelic sequences of Class II HLA genes, thereby providing complete HLAClass II genotype information for a subject. Using the method of thepresent invention complete Class II HLA typing (DR, DQ and DP) can beperformed in about 16 to 24 hours or less.

Generally, the method of the present invention involves: extraction ofsample nucleic acid; in the case of RNA, generation of cDNA; cDNA orgenomic DNA amplification; direct sequencing of amplification products;and analysis of the direct sequence information. Generation of cDNA,amplifying the cDNA and direct sequencing the cDNA amplificationproducts is accomplished using oligonucleotide primers with specificcharacteristics, such as those described herein.

A. Oligonucleotide Primers

The oligonucleotide primers of the present invention can be synthesizedusing any known suitable method, such as phosphotriester andphosphodiester methods. Narang et al., Methods Enzymol., 68:90 (1979);Brown et al., Methods Enzymol., 68:109 (1979). Oligonucleotides can beprepared using a modified solid support such as a Biosearch 8750 DNAsynthesizer. Useful primers can also be isolated from a biologicalsource using appropriate restriction endonucleases which cut doublestranded DNA at or near a nucleotide sequence of interest for use as aprimer.

B. Extraction of Sample Nucleic Acid

In the process of the present invention any source of nucleic acid canbe used as the sample nucleic acid, as long as the sample contains thenucleic acid sequence of interest. For example, the sample chosen forthe present method can be RNA, DNA or a DNA/RNA hybrid. Typical samplesinclude peripheral blood mononuclear cells, (PBMNC's), lymphoblastoidcell lines (LCL's), hair cells or the like. For determining human HLAClass II and Class I gene polymorphisms LCL's or PBMNC's are preferred.The nucleic acid to be isolated (e.g. RNA or DNA) will depend on thesource of genetic material (blood stain, hair, or peripheral bloodcells). However, in the case of HLA Class II genes including DRB1-5,DQB1, DQA1, DPA1, DPB1 the preferred isolated nucleic acid is totalcellular RNA when the typing is to be done for transplantation purposesor paternity testing. For forensic uses, genomic DNA may be thepreferred genetic material in which case different primer considerationswould be used. Cytoplasmic and poly(A)+RNA can also be used. It isenvisioned that isolation of sample nucleic acid for the present processcan be automated using a DNA/RNA extractor (such as Model 341 DNAextractor available from Applied Biosystems, Inc.; Foster City, Calif.).

C. Generation of cDNA

Complementary DNA (cDNA) of the sample nucleic acid is generated usingspecific oligonucleotide primers and cloned reverse transcriptasefollowing general conditions suggested by the enzyme manufacturer(Bethesda Research Laboratories, Gaithersburg, Md.). Specificdifferences in type and amount of primers used, dNTP concentrations andelongation times will be readily apparent to those of skill in the artbased on the Examples that follow.

D. Polymerase Chain Reaction

Amplification of cDNA or genomic DNA for each gene locus of interest isaccomplished using the polymerase chain reaction (PCR) as generallydescribed in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis. The PCRconsists of many repetitions of a cycle which consists of: (a) adenaturation step, which melts both strands of a DNA molecule; (b) anannealing step, which is aimed at allowing the primers to annealspecifically to the melted strands of the DNA molecule; and (c) anextension step, which incorporates to the primers deoxyribonucleotidescomplementary to those of the strand of DNA to which the primers areannealed. The PCR process, as indicated in the Examples, can beconducted using a Thermocycler (Perkin-Elmer, Cetus, Emeryville,Calif.).

The present invention introduces the use of non-conservedoligonucleotides in the PCR procedure specifically designed to solve theproblems associated with, for example, detecting, evaluating, and/orcharacterizing polymorphism at a polymorphic gene locus or loci of anindividual. In the case of HLA typing, the use of non-conservedoligonucleotides addresses the problems one would face in performing HLAtyping by sequencing DNA amplified exclusively by using conservedoligonucleotides or allele specific oligonucleotides (see below).

It is understood that the PCR process is designed for the amplificationof specific genes with the use of oligonucleotides specific for theparticular gene to be amplified. However, even using completely matchedprimers, in most cases the PCR is not absolutely specific. In the caseof HLA typing, for HLA-DRB genes and Class I genes, the use of conservedprimers in PCR will generate complex mixtures of templates, which upondirect sequencing will be seen as overlapping sequencing ladders,cumbersome to interpret. Therefore, genes for which the exact nucleotidesequence information is unknown can not be achieved with an adequatelevel of certainty. Use of non-conserved oligonucleotides which canselectively anneal under high stringency conditions to two or feweralleles of a gene locus or group of homologous loci can provide sequenceinformation for the different genes at highly homologous loci in complexheterozygote combinations. Thus, the present invention provides a methoduseful for determining the genotype for polymorphic gene loci. This isof particular importance to HLA typing, and is applicable to Class I HLAtyping as well as Class II typing.

The difference between non-conserved primers and allele-specificoligonucleotides resides in that the latter can only be used when thepresence of a particular allele is known, and also requires the use of aspecific primer for each of the alleles of the polymorphic system. Thus,combining use of a non-conserved primer and conserved primers to amplifythe separate alleles of highly homologous polymorphic gene loci canprovide simpler DNA polymerase chain reaction product combinationssufficient to allow unambiguous interpretation of direct sequencingladders of each allele for genotype determinations with moderateexpenditure of time and economical cost.

The conditions used for the PCR reactions are preferably the same exceptfor the temperature used in the annealing step, which is differentdepending on the type of primer used, conserved (Type 1) ornon-conserved (Type 2). Reactions that use the former primer type arepreferably performed at 37° C. in the annealing step of the cycle,whereas this step is preferably performed at about 55° C. to 60° C. inreactions that use the later type of primers. The concentrations ofprimers, and buffers used will be apparent from and include the processparameters described in the Examples that follow.

E. Direct Sequencing Of PCR Products

Direct sequencing of double-stranded DNA generated by the PCR isaccomplished using an enzyme appropriate to DNA sequencing, such as Taqpolymerase, or the like, and specific combinations of reagents atappropriate concentrations. The sequencing procedure can be conducted inan automatic sequencing apparatus such as the 373A Model DNA Sequencerfrom Applied Biosystems Inc. (Foster City, Calif.). The reagents,including sequencing primers and nucleic acid termination mixtures willbe understood by those of skill in the art based on the directsequencing procedure specified in the following Examples.

F. Analysis Of Direct Sequenced PCR Products

The nucleic acid ladders resulting from direct sequencing the cDNA orgenomic DNA for each gene locus of interest can be assessed visuallyfrom autoradiograms or by employing a computer programmed withnucleotides sequence information for all alleles of all haplotypes andprocedures for comparing sequenced alleles and known alleles of geneloci of interest. In a preferred embodiment of the present invention,the evaluation of gene locus alleles involves a two step process: (a)comparison of the gene sequences of each polymerase chain reactionproduct (i.e., conserved and non-conserved primer products) with alibrary of known genotype information such as the information obtainedon homologous cell lines very well characterized by methods other thansequencing [Marsh and Bodmer, Immunogenetics, 31:131 (1990)] as well assequences of individual alleles; followed by (b) comparison of directsequence information for the polymerase chain reaction product of anallele of a gene locus amplified with a conserved oligonucleotide primerpair and polymerase chain reaction product of alleles of a gene locus orloci amplified with a conserved/non-conserved primer pair. Thiscomparison employs a substitution algorithm or visual cancellation ofduplicative sequence ladder information to generate the specificsequence information for each allele of a gene locus.

It is envisioned that the process of the present invention can be usedto amplify and sequence known and unknown highly polymorphic systems(e.g., genetic disease-related genes, cancer-related genes, and HLAtyping, including Class I, Class II, and Class III HLA typing, and thelike). The present process is believed to be useful for paternitytesting and forensic medicine, with more accuracy than restrictionfragment length polymorphism (RFLP), DNA fingerprinting or dotblot-detection systems. While in the latter only a hybridization patternis observed, direct sequencing of amplified products shows the exactnucleotide sequence of the amplified genes, and hence is more accurateand reliable.

The method is particularly well suited for Class II HLA typing, reducingits costs, increasing its speed and especially improving its accuracy.As evidenced by the following Examples, sequence polymorphism analysisof DRB1, DRB3, DRB4, DRB5, DQB1, DQA1, DPA1 and DPB1 genes can berapidly performed in any subject of unknown HLA type by means ofenzymatic amplification and direct sequencing of Class II genes using alimited number of conserved and non-conserved oligonucleotides. Theapproach described herein is entirely automatable using currentlyavailable technology and, as opposed to previously described methodsusing oligonucleotide probes and dot blots, has the advantage ofdetecting the presence of new allelic sequences or sequencemicroheterogeneity at the population level. The methodology of thepresent invention is envisioned to be useful for detailed analyses ofthe effects of sequence allelism at different Class II HLA loci on graftsurvival after allogeneic transplantation. The method of the presentinvention allows rapid and precise sequence analysis of Class II HLApolymorphism in studies of human disease and may be of interest in thesearch for new Class II sequence variants in large populations ofsubjects.

The present invention is further described by illustration in thefollowing Examples which are not intended to limit the invention.

EXAMPLE I

1. Preparation of Oligodeoxyribonucleotide Primers and Sequence PrimerCombinations Useful for cDNA/PCR/Sequencing Reactions of Class II HLAGenes

All of the oligodeoxyribonucleotide primers described herewithin weresynthesized as described below:

Automated Synthesis of oligodeoxyribonucleotide primers: Theb-cyanoethylphosphoamidites, obtained from Milligen-Biosearch (Novato,Calif.), were sequentially condensed to a nucleoside derivatizedcontrolled pore glass support using a Biosearch 8750 DNA synthesizer.Condensation cycles included detritylation with dichloroacetic acid indichloromethane, followed by condensation with benzotriazole and cappingwith acetic anhydride and 1-methylimidazole in tetrahydrofuran andpyridine, with each cycle time being approximately 9 minutes. Yields ateach step were >99% as determined by measuring dimethoxytrityle alcoholrelease. The methodology for oligodeoxyribonucleotide synthesis isdescribed in Caruthers, et al., Methods Enzymol., 154:287 (1987).

Deprotection and purification of oligodeoxyribonucleotide primers:Deprotection and purification of oligodeoxyribonucleotide primers wasperformed using the procedure described by Schulhof et al., Nucl. AcidsRes., 15:397 (1987). Briefly, the oligodeoxyribonucleotide was removedfrom the solid support by exposure to concentrated ammonium hydroxide atroom temperature for about one hour. The solution containing thepartially deprotected oligodeoxyribonucleotide was brought to 65° C. for16 hours. Ammonia was removed and the residue was subjected tochromatography on a C18 reverse-phase column (RP 304, BioRad, Richmond,Va.) using a linear gradient of 14 to 20% acetonitrile in 0.1 molarammonium/triethylamine, pH 7.0. The dimethoxytrityle group was removedfrom the HPLC-purified oligodeoxyribonucleotide by treatment with 70%acetic acid. The detritylated oligodeoxyribonucleotide was recovered byprecipitation in ether, vacuum centrifuged until dry, resuspended inwater and quantitated by measuring its absorbance at 260 nm.

Using the above procedure, the following oligonucleotide primerscorresponding to specified regions of HLA Class II DQA, DQB, DRB, DPBand DPA loci were synthesized (see Table I below) and extensivelytested:

                                      TABLE I                                     __________________________________________________________________________    Oligonucleotides Used For The cDNA/PCR/Sequencing Reactions.                  __________________________________________________________________________    Sequence                                                                      Listing                                                                            (Seq.)                                                                   No.  Type 1                     Anneal  Locus(i)                                                                              Template                                                                              Size                  __________________________________________________________________________     1   DQB7 5'-GGTGGTTGAGGGCCTCTGTCC-3'                                                                         105-111 DQB1    RNA     RT/PCR                 2   DRB20                                                                              5'-GTGCTGCAGGGGCTGGGTCTT-3'                                                                         105-111 DRB1/3/4/5                                                                            RNA     RT/PCR                 3   DQA9 5'-GGTGAGGTTACTGATCTTGAAG-3'                                                                        148-155 DQA1    RNA     RT/PCR                 4   DQB13                                                                              5'-AGAGACTCTCCCGAGGATTTC-3'                                                                         1-7     DQB1    RNA     PCR/SEQ                5   DRB22                                                                              5'-CTGGCTTTGGCTGGGGACACC-3'                                                                         -4-3    DRB1/3/4/5                                                                            RNA/DNA PCR/SEQ                6   DRB11                                                                              5'-TGTTCTCCAGCATGGTGTGTC-3'                                                                         -33/-26 DRB1/3/4/5                                                                            RNA     PCR                    7   DQA10                                                                              5'-CTGTCCTCCGTGATGAGCCC-3'                                                                          -10/-4  DQA1    RNA     PCR                    8   DQB932                                                                             5'-TCGCCTCTGCAGGGTCGCGCG-3'                                                                         88-94   DQB1    DNA     PCR                    9   DQB931                                                                             5'-TTTAAGGGCATGTGCTACTTC-3'                                                                         11-17   DQB1    DNA     PCR                   10   DQB30                                                                              5'-ATGGGGAGATGGTCACTGTGG-3'                                                                         97-104  DQB1    RNA     SEQ                   11   DRB30                                                                              5'-AGGATACACAGTCACCTTAGG-3'                                                                         97-103  DRB1/3/4/5                                                                            RNA     SEQ                   12   DQB5 5'-GTAGTTGTGTCTGCACAC-3'                                                                            78-83   DQB1    RNA/DNA SEQ                   13   DRB12                                                                              5'-GCCGCTGCACTGTGAAGCTC-3'                                                                          87-94   DRB1/3/4/5                                                                            RNA     SEQ                   14   DQA29                                                                              5'-CACGGTTCCGGTAGCAGCGGTAG-3'                                                                       82-89   DQA1    RNA     SEQ                   15   DQA30                                                                              5'-TACGGTCCCTCTGGCCAG-3'                                                                            19-24   DQA1    RNA     SEQ                   16   DRB1400                                                                            5'-GCGCTTCGACAGCGACGTGG-3'                                                                          38-45   DRB1/3/4/5                                                                            RNA/DNA SEQ                   17   DRB1401                                                                            5'-GAGGTGACTGTGTATCCTGAC-3'                                                                         98-104  DRB1*0701-2                                                                           RNA/DNA PCR                   18   DRB1402                                                                            5'-GATCAGGCCTGTGGACACCAC-3'                                                                         142-148 DRB1/3/4/5                                                                            RNA/DNA PCR                   19   DRB1403                                                                            5'-CCGGAACCACCTGACTTCAAT-3'                                                                         127-133 DRB1/3/4/5                                                                            RNA/DNA SEQ                   20   DRB1406                                                                            5'-GCCAAGAGTGGGCCTCGCAGC-3'                                                                         bp 18-38-                                                                             DRB1/3/4/5                                                                            DNA     PCR                                                   intron 3                                      21   DRB825                                                                             5'-AACCCCGTAGTTGTGTCTGCA-3'                                                                         79-85   DRB1/3/4/5                                                                            DNA     SEQ                   22   DRB824                                                                             5'-GGGGACACCCGACCACGTTTC-3'                                                                         1-7     DRB1/3/4/5                                                                            DNA     PCR                   23   DPB10                                                                              5'-CGGACAGTGGCTCTGACGGCG-3'                                                                         -19/-13 DPB1    RNA     PCR                   24   DPB11                                                                              5'-GTTGTGGTGCTGCAAGGGCCC-3'                                                                         105-111 DPB1    RNA     RT/PCR                25   DPB12                                                                              5'-CTTGGAGGGGGAAACATTCAC-3'                                                                         97-103  DPB1    RNA     SEQ/RT                26   DPB13                                                                              5'-TACTGATGGTGCTGCTCACAT-3'                                                                         -12/-5  DPB1    RNA     SEQ                   27   DPB14                                                                              5'-AGAGGGAGAAAGAGGATTAGA-3'                                                                         bp -42/-62                                                                            DPB1    DNA     PCR                                                   intron 2                                      28   DPB15                                                                              5'-GCCCTGGGCACGGGCCCGCGG-3'                                                                         bp 39/59                                                                              DPB1    DNA     PCR                                                   intron 3                                      29   DPB16                                                                              5'-CGGCCCAAAGCCCTCACTCAC-3'                                                                         bp 1-21 DPB1    DNA     SEQ                                                   intron 3                                      30   DPB17                                                                              5'-CGCTCATGTCCGCCCCCTCCC-3'                                                                         bp -6/-26                                                                             DPB1    DNA     SEQ                                                   intron 2                                      31   DPA14                                                                              5'-GTCAATGTGGCAGATGAGGGT-3'                                                                         104-110 DPA1    RNA     RT/PCR                32   DPA15                                                                              5'-CATATCAGAGCTGTGATCTTG-3'                                                                         -17/-23 DPA1    RNA     PCR                   33   DPA16                                                                              5'-CTTGGGAAACACGGTCACCTC-3'                                                                         88-94   DPA1    RNA     SEQ                   34   DPA17                                                                              5'-CTGCTGAGTCTCCGAGGAGCT-3'                                                                         -3/-9   DPA1    RNA     SEQ                   35   DPA10                                                                              5'-CTCTAGCTTTGACCACTTGC-3'                                                                          bp -69 to                                                                             DPA1    DNA     PCR                                                   -50 intron 2                                  36   DPA11                                                                              5'-AGTCTGAGGGTGGCAGAGAGG-3'                                                                         bp 55-71                                                                              DPA1    DNA     PCR                                                   intron 3                                      37   DPA12                                                                              5'-GGCCTGAGTGTGGTTGGAACG-3'                                                                         76-82   DPA1    DNA/RNA SEQ                   38   DPA18                                                                              5'-CTGGCTAACATTGCTATATTG-3'                                                                         59-65   DPA1    RNA     PCR                   39   DPA19                                                                              5'-GGTCCCCTGGGCCCGGGGGTC-3'                                                                         222-228 DPA1    RNA     RT                    40   DPA20                                                                              5'-GCCAGAACGCAGAGACTTTAT-3'                                                                         214-220 DPA1    RNA     SEQ                   41   DPA21                                                                              5'-AACTTGAATACCTTGATCCAG-3'                                                                         68-74   DPA1    RNA     SEQ                   __________________________________________________________________________    Sequence                                                                      Listing                                                                            (Seq.)                                                                   No.  Type 2                                                                             Anneal                Locus(i)                                                                              Template                                                                              Step                          __________________________________________________________________________    42   DRB23                                                                              5'-TTCTTGCAGCAGGATAAGTA-3'                                                                          7-13    DRB1    RNA/DNA PCR                   43   DRB24                                                                              5'-CCACGTTTCTTGGAGTACTCT-3'                                                                         5-11    DRB1    RNA/DNA PCR                   44   DRB25                                                                              5'-TTTCTTGGAGCAGGTTAAACA-3'                                                                         6-13    DRB1    RNA/DNA PCR                   45   DRB16                                                                              5'-AGATGCATCTATAACCAAGAG-3'                                                                         29-35   DRB1/3/4/5                                                                            RNA/DNA PCR                   46   DRB17                                                                              5'-AGATACTTCCATAACCAGGAG-3'                                                                         29-35   DRB1/3/4/5                                                                            RNA/DNA PCR                   47   *DQB6                                                                              5'-CTGAGCACCCCAGTGGCTGAG-3'                                                                         -8/-2   DQB1    RNA     PCR                   48   *DQB14                                                                             5'-CTGAGCTCCTCACTGGCTGAG-3'                                                                         -8/-2   DQB1    RNA     PCR                   49   *DQB15                                                                             5'-CTGAGCACCTCGGTGGCTGAG-3'                                                                         -8/-2   DQB1    RNA     PCR                   __________________________________________________________________________     All the above Type 1 primers are annealed at 37° C. and the Type 2     primers are annealed at 55° C. When the latter anneal at 37.degree     C. in the PCR, they do not distinguish among allelic transcripts differin     by few base pairs. This list of primers includes primers which are only       used in certain situations, such as to confirm homozygosity at a              particular locus whenever not expected according to the typings performed     at the other linked loci. The alternative combinations of primers used in     each step are described in Table II below. [(*) These primers anneal to a     polymorphic region of DQB1 cDNAs (codons -8 to -2) encoding the 3' end of     the signal peptide which has specific nucleotide nucleotide sequences for     different DQB1 alleles (DQB6DQB1*0601 and DQB1*0604, DQB14DQB1*0501-,         DQB15DQB1*0301-).] RT = Reverse transcription; SEQ = sequencing.         

2. Combinations of Primers for cDNA/PCR/Sequence Reactions

There are specific combinations of oligonucleotide primers for eachreaction and for each locus, including cDNA synthesis, PCR amplificationand direct sequencing, which are designed to provide all the necessarysequence information for obtaining highly accurate, fast and inexpensivetyping results. These combinations are listed in Table II below as"routine" combinations. In addition, Table II includes a list of"alternative" combinations of oligonucleotides for each locus which maybe used to confirm results obtained with the "routine" combinations fora particular locus not expected according to, for instance, knownhaplotypic maps. These "unexpected" results are usually indicative ofthe existence of new alleles and/or haplotypes, which can be confirmedwith the use of the alternative combinations of oligonucleotides. In anycase, each of these combinations of oligonucleotides is characterized byits ability to generate an end-product (sequencing ladder) which issuitable of being accurately read by the naked eye or processed bycomputer operated under appropriate software.

For typing purposes in the clinical setting, such as in transplantation,the method uses RNA isolated from peripheral blood mononuclear cells asstarting material; for forensic purposes, however, DNA is often the onlyavailable template. Although for each template (RNA or DNA) differentcombinations of oligonucleotides are used (see Table II), the generalstrategy for typing, including the interpretation of the results isessentially the same. The specific combinations of primers for "routine"RNA and DNA analysis, respectively, are described below in more detail.The general overview of the HLA typing strategy is shown in FIGS. 1 and2.

                                      TABLE II                                    __________________________________________________________________________    Combinations of Primers for cDNA/PCR/Seg Reactions                            __________________________________________________________________________    1. RNA                                                                        Type  cDNA   PCR    A.T.                                                                              Seq*                                                  __________________________________________________________________________    Routine                                                                       A. 1  DRB20(2)                                                                             DRB11(6)                                                                             37° C.                                                                     DRB30/DRB12(13)/DRB22**(5)                            B. 2  DRB20(2)                                                                             DRB23(42)                                                                            55° C.                                                                     DRB30(12)/DRB12(13)                                   C. 2  DRB20(2)                                                                             DRB24(43)                                                                            55° C.                                                                     DRB30(11)/DRB12(23)                                   D. 2  DRB20(2)                                                                             DRB25(44)                                                                            55° C.                                                                     DRB30(12)/DRB12(13)                                   E. 1  DQB7(1)                                                                              DQB13(4)                                                                             37° C.                                                                     DQB30(14)/DQB5(12)                                    F. 1  DQA9(3)                                                                              DQA10(7)                                                                             37° C.                                                                     DQA30(15)/DQA29***(14)                                G. 1  DPB11(24)                                                                            DPB10(23)                                                                            37° C.                                                                     DPB12(25)/DPB13(26)                                   H. 1  DPA14(31)                                                                            DPA15(32)                                                                            37° C.                                                                     DPA16(33)/DPA17(34)                                   I. 1  DPA19(39)                                                                            DPA18(38)                                                                            37° C.                                                                     DPA20(40)/DPA21****(41)                               Alternative                                                                   J. 2  DRB20(2)                                                                             DRB16(45)                                                                            55° C.                                                                     DRB30(11)/DRB12(13)                                   K. 2  DRB20(2)                                                                             DRB17(46)                                                                            55° C.                                                                     DRB30(11)/DRB12(13)                                   L. 1  DRB20(2)                                                                             DRB22(5)                                                                             37° C.                                                                     DRB30(11)/DRB12(13)                                   M. 1  DQB7(1)                                                                              DQB6(47)                                                                             37° C.                                                                     DQB13(4)                                              N. 2  DQB7(1)                                                                              DQB6(47)                                                                             55° C.                                                                     DQB30(10)/DQB5(12)                                    O. 2  DQB7(1)                                                                              DQB14(48)                                                                            55° C.                                                                     DQB30(10)/DQB5(12)                                    P. 2  DQB7(1)                                                                              DQB15(49)                                                                            55° C.                                                                     DQB30(10)/DQB5(12)                                    Q. 1  DPB12(25)                                                                            DPB10(23)                                                                            37° C.                                                                     DPB13(26)                                             R. 1  DPA16(33)                                                                            DPA15(32)                                                                            37° C.                                                                     DPA17(34)                                             __________________________________________________________________________    2. DNA                                                                        Type  PCR1   PCR2   A.T.                                                                              Seq                                                   __________________________________________________________________________    Routine                                                                       S. 1  DRB1406(20)                                                                          DRB22(8)                                                                             37° C.                                                                     DRB12(13)/DRB1400*****(16)                            T. 2  DRB1406(20)                                                                          DRB24(43)                                                                            55° C.                                                                     DRB12(13)/DRB1400(16)                                 U. 2  DRB1406(20)                                                                          DRB25(44)                                                                            55° C.                                                                     DRB12(13)/DRB1400(16)                                 V. 2  DRB1406(20)                                                                          DRB23(42)                                                                            55° C.                                                                     DRB12(13)/DRB1400(16)                                 W. 1  DQB932(8)                                                                            DQB931(9)                                                                            37° C.                                                                     DQB5(12)                                              X. 1  DPB14(27)                                                                            DPB15(28)                                                                            37° C.                                                                     DPB16(29)/DPB17(30)                                   Y. 1  DPA10(35)                                                                            DPA11(36)                                                                            37° C.                                                                     DPA12(37)                                             Alternative                                                                   Z. 1  DRB12(13)                                                                            DRB824(22)                                                                           37° C.                                                                     DRB825(21)                                            AA.                                                                              2  DRB1401(17)                                                                          DRB1402(18)                                                                          55° C.                                                                     DRB1403#(19)                                          AB.                                                                              2  DRB1406(20)                                                                          DRB16(45)                                                                            55° C.                                                                     DRB825(21)/DRB12(13)                                  AC.                                                                              2  DRB1406(20)                                                                          DRB17(46)                                                                            55° C.                                                                     DRB825(21)/DRB12(13)                                  AD.                                                                              1  DPB14(27)                                                                            DPB16(29)                                                                            37° C.                                                                     DPB17(30)                                             __________________________________________________________________________     (*)For sequencing DRB and DQB two alternative sequencing primers are          indicated, both sequencing the positive strand of DNA.                        (**)Primer DRB22 is used to sequence the negative strand whenever new         allelic sequences are identified.                                             (***)Each DQA1 sequencing primer anneals to a different strand. Reaction      uses an alternative amplification primer (DRB 22 instead of DRB11) in         hypothetical situations where homozygosity may not be expected according      to the rest of the haplotype. Reaction M is used for sequencing the           negative strand of DQB1 in situations where new allelic sequences are         identified.                                                                   (****)Sequencing of the third exon is necessary to distinguish among          certain DPA1 alleles.                                                         (*****)Primer DRB1400 may be used in sequencing amplified DRB genes from      genomic DNA to read the sequences immediately following the 3'                amplification primer. Reaction Q, R, Z and AD are alternative combination     for confirming homozygosity at the corresponding loci which may not be        expected according to the rest of the Class II haplotype.                     (#)This primer combination is used to distinguish between DRB1*0701 and       DRB1*0702, which differ by a single base pair in their third exons.           () The sequence identification number (SEQ ID NO.) is in parenthesis          immediately following each primer designation.                           

EXAMPLE II Protocol: HLA Class II "Typing" by Direct Sequencing of DRB,DQB, DQA, DPA and DPB Genes

1. Cell Lines and Subjects

Lymphoblastoid cell lines (LCLs) representing each of the known Class IIhaplotypes defined at the 10th International Histocompatibility Workshop[Dupont, Hum. Immunol., 26, 3 (1989)] were provided by Dr. Miriam Segall(University of Minnesota). Forty unrelated subjects who had beenpreviously serologically typed for Class I and Class II antigens werealso studied. The serological types of each of the subjects under studywere not known to the investigator performing the sequence analysis atthe time the analysis was performed. These subjects included bothhealthy and affected (insulin-dependent diabetes and autoimmune thyroiddisease) individuals. The sequenced haplotypes, many in heterozygotecombinations, included: DR7 (n=3), DRw17 (n=26), DR4 (n=16), DRw11(n=8), DRw8 (n=4), DR1 (n=6), DRw15 (n=6), DRw16 (n=2), DRw13 (n=2),DRw14 (n=2), DR212 (n=3), DR5×6 (n=3). The cell lines and heterozygotecombinations tested are shown in Table III. Since the complexity at DPAand DPB loci is similar to that of DQ genes, the primer combinations forDPA and DPB typing were optimized in a smaller group of homozygote andheterozygote subjects.

2. HLA-DRB, DQB and DQA Transcript Amplification Using Conserved andNon-Conserved Oligonucleotides

Total cellular RNA was prepared from (1 μg) from 5-10×10⁶ peripheralblood mononuclear cells (PBMNC) or lymphoblastoid cell lines (LCLs) bycesium chloride centrifugation [Chirgwin et al. Biochemistry, 18, 5249(1979)]. Alternatively, total RNA from peripheral blood (2-10 ml) waspartially purified using a much faster protocol [Gouuh, Anal. Biochem.,173, 93 (1988)]. One microgram of total cellular RNA was reversetranscribed with Moloney leukemia virus reverse transcriptase (MLVRT)(200 u, Bethesda Research Laboratories) in 50 mM Tris HCl, pH 8.3, 75 mMKCl, 10 mM DTT, 3mM MgCl₂, in the presence of the ribonuclease inhibitorRNAs in (10 units, Promega), 75 μM each dNTP and 10 pmols of a specificnon-sense primer (Table II) in a 20 ml final volume for 30-45 min at 37°C. Eight μl of 10X PCR buffer (500 mM KCl, 100 mM Tris-Cl, pH 8.3,7.5-15 mMMgCl₂, 0.1% gelatin) were added after the incubation period. A5'-primer (20 pmols) (Type 1 or Type 2 primers, respectively, see TableII) plus 10 pmols of the non-sense primer and two units of Taqpolymerase were also added and the final volume was adjusted to 100 μlwith distilled water. The reaction mixture was subjected to 35 cycles of30 sec at 94° C., 30 sec at 37° C. or 55° C. and 30 sec at 72° C. usinga Perkin-Elmer Cetus Thermocycler [see Saiki et al., supra (1985);Mullis and Faloona, supra (1987); Saiki et al., supra (1986); Scharf etal., supra (1986)]. The primers used here, their corresponding sequencesand the regions to which they anneal are shown in Table II. Thereactions for each locus are usually performed in separate microfugetubes. However, when using conserved primers, the cDNA and PCR reactionsfor all loci (DRB, DQA, DQB, DPA and DPB) can be successfully performedsimultaneously in the same tube.

3. Direct Sequencing of Amplified Products with Taq Polymerase

The reaction mixture (100 μl ) was freed of unincorporated dNTPs andexcess of oligonucleotides by spin-dialysis using Centricon-100 (Amicon)or Ultrafree-100 (millipore) microconcentrators. One half of theretentate (20 μl) was dried down and resuspended in 15 μl of 1X Taqsequencing buffer (50 mM Tris-HCl, pH 9, 10 mM MgCl2). Internaloligonucleotides were used for priming the sequencing of DQB, DRB, DQA,DPB and DPA genes, respectively (Table II). Primers for sequencing eachstrand are listed in Table II. Only one strand is routinely sequencedfor typing; sequencing of the other strand is performed in cases where anew allelic sequence is suspected. Eighty to 100 ng of primer wereend-labelled with 10 pmol of gamma-P32 labelled ATP (5000 Ci/mmol, 10μCI/μL) and 5 units of T4 polynucleotide kinase (Promega Biotec) in a 10μl final volume. Ten ng of primer (1 μl) were added to the sequencingmixture without extraction of unincorporated labelled ATP, boiled for 5min., and then left at room temperature for 15 min. Eight units ofrecombinant Taq polymerase (USB) were added to the mixture. Four μl ofthe annealed primer/template mixture were later added to 4 μl of each ofthe stop nucleotide mixes: a) Term mix ddG: 15 microM each dGTP, dATP,dCTP, dTTP; 45 microM ddGTP; b) Term mix ddA: 15 microM each dGTP, dATP,dCTP, dTTP; 600 microM ddATP; c) Term mix ddT: 15 microM each dGTP,dATP, dCTP, dTTP; 1200 microM ddCTP; d) Term mix ddC: 15 microM eachdGTP, dATP, dCTP, dTTP; 450 microM ddCTP. The reactions were allowed toproceed for two consecutive periods of 10 min. at 72°-74° C. After thesecond cycle, each reaction was chased with 2 μl of a 7.5 μM mixture ofATP, GTP, TTP, CTP, and allowed to proceed for 5 min. After spinningdown, the reaction was stopped by adding 4 ml of 95% (vol/vol)formamide/20 mM EDTA, heated to 80° C. for 5 min. and loaded on a 0.4 mmthick 6% polyacrylamide/7M urea gel. Electrophoresis was performed at2500 V for 2 hr, the gel fixed in 5% (vol/vol) glacial acetic acid/5%(v/v) methanol for 15 min, dried, and exposed to Kodak X-Omat film for 4to 12 hours.

RESULTS

1. Sequence-Based Typing of DR and DQ Polymorphic Genes in HomozygousTyping Cells

Homozygous lymphoblastoid cell lines (LCLs) from the panel of the 10thInternational Histocompatibility Workshop (Table III) were used as aninitial test of the methodology. In total, these cell lines wererepresentative of most of the known DR and DQ alleles at the time theanalysis was conducted.

Total cellular RNA isolated from homozygous LCLs was reverse-transcribedand the resultant cDNAs amplified using conserved oligonucleotidesspecific for DRB1/DRB3/DRB4/DRB5 or DQB1 or DQA1 genes as described inthe preceding protocol. The conserved or Type 1 oligonucleotide primersanneal to regions of conserved DNA sequences; these regions areidentical among the known alleles at each locus and flank the secondexon of Class II genes. These conserved primers, as opposed tonon-conserved or Type 2 primers, are designed to amplify all knownalleles at DRB, DQA1 and DQB1 loci and, thus, all possible combinationsof these alleles in any given heterozygote. The Type 1 oligonucleotidesdid not cross-amplify templates at loci other than those specified bythe oligonucleotides (i.e., the DQA1 primers did not amplify DRB or DQB1transcripts and vice versa); as expected, the DRB primers also amplifiedany DRB3, DRB4 or DRB5 transcripts present in addition to DRB1.Sequencing of these amplified templates was performed using a Type 1primer annealing to a conserved region of the cDNAs internal to thesequence recognized by the amplification primers. FIG. 1A shows thegeneral strategy for the method (SBT) and FIGS. 1B to 1F show therelative position of each of the oligonucleotides used for the cDNA, PCRand sequencing reactions on the mature DRB, DQA and DQB mRNA molecules.The sequences of these primers, the loci they are specific for, thespecific positions (codons) to which they anneal and the reaction(s)they are used in are indicated in Table II where the specificcombinations of primers that can be used for the cDNA/PCR/sequencingreactions for each locus are identified. As noted in the legend to TableII, some of the primer combinations shown represent alternatives whichmay be useful in confirming results for a particular locus which do notfit with the expected sequences usually found with the rest of thehaplotype. Each cDNA/PCR reaction is usually performed in a separatetube. However, when using Type 1 primers, the cDNA/PCR reactions for allthe loci (DRB, DQA, DQB, DPA and DPB) can be performed simultaneously inthe same tube. The products of each locus are sequenced in separatetubes. Following the conditions described in the above protocol, thesequence ladders between the sequencing primer and the 5' amplificationprimer could be clearly read starting from 2 to 14 bases from thesequencing primer binding site. No anomalous amplification products orsequencing ladders were detectable upon direct sequence analysis ofamplified DRB, DQB1 and DQA1 cDNAs from the 43 homozygous cell linestested (Table IIIa). The specific alleles at each Class II HLA locuscomposing the haplotypes carried by each of these cell lines are shownin Table IIIb. The number of ladders generated for each cell line wasalways that expected according to the specificity of the amplificationprimers (one DQB1 and one DQA1 ladder for all cell lines, one DRB ladderfor DR1 and DRw8 cell lines and two DRB ladders for haplotypes of theDRw52 and DRw53 supertypic groups). Thus, analysis of the homozygoustyping cell lines showed that the Type 1 primers used for cDNAsynthesis, PCR and sequencing reactions allowed for accurateamplification and sequencing of all the tested alleles at each of theseloci.

                  TABLE IIIa                                                      ______________________________________                                        Cell Lines and Heterozyqote Combinations Tested                               Cell      Class II      Sub-   Class II                                       Line      HLA Type      ject   HLA Types**                                    ______________________________________                                        SA        DR1-Dw1       S1     DR1-Dw1/DRw17                                  MZ070782  DR1-Dw20      S2     DR1-Dw1/                                                                      DR4-Dw4                                        KAS011    DRw16-Dw21    S3     DR1-Dw1/DRw8.1                                 *CALOGERO DRw16-Dw-     S4     DRw15-Dw2/                                                                    DRw17                                          *WJR076   DRw16-Dw21    S5     DRw15-Dw2/                                                                    DR4-Dw4                                        *DEM      DRw16-Dw21/DR4                                                                              S6     DRw16-Dw21/                                                                   DRw17                                          WT24      DRw16-Dw21    S7     DR5x6@/DRw17                                   RML       DRw16-Dw22    S8     *DRw13-Dw18/                                                                  DRw17                                          SCHU      DRw15-Dw2     S9     DRw13-Dw19/                                                                   DRw17                                          WT8       DRw15-Dw2     S10    DR4-Dw4/DRw17                                  *AMAI     DRw15-Dw2     S11    DR4-Dw4/DRw12                                  E4181324  DRw15-Dw12    S12    DR4-Dw13/                                                                     DR1-Dw1                                        MT14B     DR4-Dw14      S13    DR4-Dw13/DRw17                                 EJ32B     DRw17-SYD3    S14    DR4-Dw14/                                                                     DRw15-Dw2                                      RSH       DRw18-DwRSH   S15    *DR4-Dw15/                                                                    DRw17                                          DEU       DR4-Dw4       S16    DRw11-Dw5/                                                                    DRw17                                          WT51      DR4-Dw4       S17    DRw12/DR1-Dw1                                  JBAF      DR4-Dw13      S18    DRw12/DRw8.1                                   YAR       DR4-Dw10      S19    *DRw14-Dw9/                                                                   DRw17                                          KT17      DR4-DKT2      S20    DR7/DRw17                                      SPOO10    DRw11-DB2     S21    DR4-Dw4/DR7                                    JBUSH     DRw11-Dw5     S22    DRw8.1/DR7                                     TISI      DRw11-DwTISI  S23    DRw8.1/DR5x6@                                  JVM       DRw11-DwJVM   S24    DRw8.2/                                                                       DRw11-Dw5                                      BM16      DRw12-DB6     S25    DRw8.3/DR1-Dw1                                 *H0301    DRw13-Dw19    S26    DRw8.3/                                                                       DRw15-Dw2                                      WDV       DRw13-Dw18    S27    DR9/DR1-Dw1                                    WT47      DRw13-Dw19                                                          TEM       DRw14-Dw9                                                           EK        DRw14-Dw9                                                           AMALA     DRw14-Dw16                                                          LBF       DR7-DB1                                                             BH        DR7-DB1                                                             CF96      DR7-Dw7                                                             BER       DR7-Dw7                                                             DBB       DR7-Dw11                                                            MOU       DR7-Dw17                                                            BTB       DRw8-Dw8.1                                                          OLGA      DRw8-Dw8.2                                                          LUY       DRw8-Dw8.3                                                          TAB089    DRw8-Dw8.3                                                          DKB       DR9-Dw23                                                            ______________________________________                                         The allelic composition at DRB, DQA1 and DQB1 loci for the sequenced          haplotypes corresponded to that expected according to published sequence      information from well characterized homozygous cell lines unless indicate     (*).                                                                          *Haplotypes carrying new allelic sequences (DRB1, DRB3, DQA1 or DQB1          loci).                                                                        *Only the tested heterozygote combinations are listed. The remainder of       the 40 subjects tested were homozygotes or carried the haplotypes listed      in this table.                                                                @This DRB specificity (DR5x6) has been given this arbitrary designation       according to serological, RFLP and sequence information.                 

                                      TABLE IIIb                                  __________________________________________________________________________    Allelic Composition of Human Class II Haplotypes                              Haplotype WS#     DRB1 DRB3                                                                              DRB4                                                                              DRB5                                                                              DQB1 DQA1                                  __________________________________________________________________________    DR1-Dw1   9001    *0101                                                                              --  --  --  *0501                                                                              *0101                                 #DR1-Dw20 9002    *0102                                                                              --  --  --  *0501@                                                                             *0101                                 DRw16-Dw21                                                                              9009,-84,-15                                                                          *1601                                                                              --  --  *0201                                                                             *0502                                                                              *0102                                 #DRw16-Dw21                                                                             9012,9007                                                                             *1601                                                                              --  --  *0202                                                                             *0502                                                                              *0102                                 DRw16-Dw22                                                                              9016    *1602                                                                              --  --  *0202                                                                             *0301                                                                              *0501                                 DRw15-Dw2 9013,9017                                                                             *1501                                                                              --  --  *0101                                                                             *0602                                                                              *0102                                 #DRw15-Dw2                                                                              9010    *1501                                                                              --  --  -1.3                                                                              *0602                                                                              *0102                                 DRw15-Dw12                                                                              9011    *1502                                                                              --  --  *0102                                                                             *0601                                                                              *0103                                 DRw17-Dw3 9088    *0301                                                                              *0101                                                                             --  --  *0201                                                                              *1501                                 DRw17-DwSYD                                                                             9085    *0301                                                                              *0201                                                                             --  --  *0201                                                                              *0501                                 DRw18-DwRSH                                                                             9021    *0302                                                                              *0101                                                                             --  --  *0402                                                                              *0401                                 DR4-Dw4   9025    *0401                                                                              --  *0101                                                                             --  *0301                                                                              *0301                                 DR4-Dw4   9029    *0401                                                                              --  *0101                                                                             --  *0302                                                                              *0301                                 DR4-Dw13  9030    *0403                                                                              --  *0101                                                                             --  *0301                                                                              *0301                                 DR4-DKT2  9024    *0403/6                                                                            --  *0101                                                                             --  *0302                                                                              *0301                                 DR4-Dw10  9026    *0402                                                                              --  *0101                                                                             --  *0302                                                                              *0301                                 DR4-Dw14  9028    *0404                                                                              --  *0101                                                                             --  *0302                                                                              *0301                                 DR4-Dw15 KT3 (a)  *0405                                                                              --  *0101                                                                             --  *0401                                                                              *0301                                 #         CC      *0405                                                                              --  *0101                                                                             --  *0201                                                                              *0301                                 DRw11-DB2 9036    *1104                                                                              *0201                                                                             --  --  *0502                                                                              *0102                                 DRw11-Dw5 9035    *1101                                                                              *0201                                                                             --  --  *0301                                                                              *0501                                 DRw11-Dwn 9042    *1103                                                                              *0201                                                                             --  --  *0301                                                                              *0501                                 DRw11-DwJVM                                                                             9039    *1102                                                                              *0201                                                                             --  --  *0301                                                                              *0501                                 DRw12-DB6 9038    *1201                                                                              *0201                                                                             --  --  *0301                                                                              *0501                                 #         5x6     DR5x6                                                                              *0101                                                                             --  --  *0301                                                                              *0501                                 #DRw13-Dw19                                                                             9055    *1302                                                                              *0301                                                                             --  --  DQB6.5                                                                             *0102                                 DRw13-Dw18                                                                              9062    *1301                                                                              *0101                                                                             --  --  *0603                                                                              *0103                                 DRw13-Dw18 (b)    *1301                                                                              *0101                                                                             --  --  *0502                                                                              *0102                                 DRw13-Dw19                                                                              9063    *1302                                                                              *0301                                                                             --  --  *0604                                                                              *0102                                 DRw14-Dw9 9057,9054                                                                             *1401                                                                              *0201                                                                             --  --  *0503                                                                              DQA1.4                                #         MA      *1401                                                                              *0201                                                                             --  --  DQB5.4                                                                             ND                                    DRw14-Dw16                                                                              9064    *1402                                                                              *0101                                                                             --  --  *0301                                                                              *0501                                 DR7-DB1   9096,9046                                                                             *0701                                                                              --  *0101                                                                             --  *0201                                                                              *0201                                 DR7-Dw7   9094,9093                                                                             *0701                                                                              --  *0101                                                                             --  *0201                                                                              *0201                                 DR7-Dw11  9052    *0701                                                                              --  *0101&                                                                            --  *0303                                                                              *0201                                 DR7-Dw17  9050    *0701                                                                              --  *0101                                                                             --  *0201                                                                              *0201                                 DRw8-DW8.1                                                                              9067    *0801                                                                              --  --  --  *0402                                                                              *0401                                 DRw8-Dw8.2                                                                              9071    *0802                                                                              --  --  --  *0402                                                                              *0401                                 DRw8-Dw8.3                                                                              9070    *0803                                                                              --  --  --  *0301                                                                              *0601                                 DRw8-Dw8.3                                                                              9066    *0803                                                                              --  --  --  *0601                                                                              *0103                                 DR9-Dw23  9075    *0901                                                                              --  *0101                                                                             --  *0303                                                                              *0301                                 DRw10 (c-e)       *1001                                                                              --  --  --  *0501                                                                              *0101                                 __________________________________________________________________________     @, the DQB gene of the DR1Dw20 haplotype differs from that of the DR1Dw1      haplotype by a silent nucleotide substitution at codon 68. # indicates th     new haplotypes presented in this report. &, This cell line does not           translate its DRB4 mRNA (Knowles, 1989). ND, not determined. (a) Gregerse     et al. 1986; (b) Kao et al. 1989; (c) Merryman et al. 1988; (d) Todd et       al. 1987; (e) Merryman et al. 1989.                                      

2. Amplification and Direct Sequencing of DQA1 and DQB1 cDNAs inSubjects of Unknown HLA Type

DNA sequences have been determined for most HLA Class II allelicspecificities defined by conventional HLA typing techniques (March, S.G. E., Bodmer, J. G. HLA-DRB nucleotide sequences, 1990. Immunogenetics31:141, 1990; Todd, J. A., Bell, J. I., McDefvitt, H. O.: HLA-DQB genecontributes to susceptibility and resistance to insulin-dependentdiabetes mellitus. Nature 329:599, 1987). Comparisons of these sequencesindicates that any given DQA1 or DQB1 homozygous or heterozygous alleliccombination is characterized by a specific sequencing ladder.

Total RNA from PBMNCs from 40 different subjects was tested to evaluateif the allelic composition of DQA1 and DQB1 homo- and heterozygotescould be determined correctly by direct amplification and sequencingusing Type 1 primers. These subjects had been previously serologicallytyped but the typing information was not known to the investigator whoassigned the Class II allelic specificities from the sequencing results.These 40 subjects comprised 27 different heterozygote combinations(Table III). All individuals were assigned DQA1 and DQB1 allelicsequences that were consistent with the serological phenotypes. In allthe heterozygotes tested, both allelic sequences could be read clearlyfrom the composite sequence pattern. A unique pattern is found for everyparticular heterozygote combination in the same way that certain RFLPbanding patterns correspond to certain heterozygote alleliccombinations. For instance, in a DQB2/DQB1.1 heterozygote one would findthe sequence GGGG(A/T)T(T/A)CCGGGC(A/G) at codons 45 to 49 which canonly be attributed to that particular allele combination. In practice,interpretation of heterozygous sequence ladders is initiated by readingcertain polymorphic positions where allele-specific bases may be found,such as, for instance, the second base of codon 46, where DQB1*0201 isthe only allele that has an A. The sequences of the two possibletemplates are then deduced and compared with the sequences of all knownalleles at the different loci.

The absence of expected bands or the presence of unexpected bands for aparticular allele or allelic combination is therefore suggestive ofsequence heterogeneity, i.e., new alleles. The same can be said for DPA1and DPB1 typing when appropriate primer combinations are used (TableIII). For instance, substitution of the A at the second base of codon 46would strongly suggest the presence of a sequence variant of DQB1*0201.Once detected, the sequence of the variant can be confirmed afterselective amplification of the variant or by subcloning the amplifiedproducts.

3. Amplification and Direct Sequencing of DRB cDNAs From Subjects ofUnknown HLA Type

As described above, the use of Type 1 primers allows the unambiguoussequencing of all heterozygous combinations of DQA1 and DQB1 alleles.The same can be said for DPA1 and DPB1 typing when appropriate primercombinations are used (Table II). Because of the isotypic complexity ofDRB genes (expression of more than one DRB locus by certain haplotypes),amplification and sequencing of cDNAs from DRB heterozygotes with Type 1primers can generate up to four overlapping ladders, thus generatingcomplex sequencing patterns.

DRB cDNAs from the same 40 individual tested above for DQA1 and DQB1genes were amplified and sequenced using DRB-specific Type 1 primers. Asmentioned above, these 40 individuals comprised 27 differentheterozygote combinations, including several examples from each of thegroups of complex DRB allelic combinations which would generate up tofour sequencing ladders. The DRB sequence ladders generated with Type 1primers were analyzed as described above for DQA1 and DQB1 loci: highlypolymorphic positions were analyzed first for the presence of bandsunique to specific alleles or groups of alleles (i.e., DR4) and thesequences deduced and compared with the sequences of all known allelesat all loci. For all but one sample, the information deduced from thesesequencing experiments matched the independently determined serologicalphenotypes of the subject under study as well as the DQA1 and DQB1allelic types assigned to these individuals by direct sequencing ofthese genes as described above. The inconsistent sample had beenserologically typed as DRw13/DR4 but was typed by sequence analysis asDRw13/DRw8-Dw8.1. The presence of a DRB1*0801 allele instead of aDRB1*0401 allele was confirmed in a repeated experiment; we thus believethat the serological typing was in error. In all the 40 cases, all DQB1,DQA1 and DRB1 templates had been equally amplified and sequenced with asimilar efficiency by the use of Type 1 primers. DRB3, DRB4 and DRB5sequence ladders could be read in all but one case (a DRB3*0101 [DRw52a]sequence was not initially observed in a DRw13/DRw17 heterozygote).Since DRB3*0101 is in linkage disequilibrium with DRB1*0301, the formerallele was expected to be found in the overlapping ladder as well. Inorder to rule out the possibility of an error, the investigatorassigning the HLA types from the sequencing ladders repeated the typingof this individual; the DRB3*0101 could be read in the repeatedexperiment.

Although the results generated by the use of Type 1 primers werecompatible with the serological phenotypes, the exclusive use of Type 1primers will not allow in all cases to assign each of the specificladders to each of the expressed loci in all possible heterozygotes.Given below are the most complex situations which cannot be addressed bythe exclusive use of Type 1 primers: 1) distinction among the differentDR4 allelic sequences in certain heterozygotes since they differ by onlya few nucleotide base pairs and such differences could be masked by thepresence of additional ladders; 2) to distinguish between DRB1*1601 andDRB1*1502 since their sequence differences will be masked by those oftheir linked DRB5 alleles; 3) to distinguish between DRB1*1301 andDRB1*1302 (which only differ at codon 86 since this difference can alsobe masked by other ladders; and finally 4) distinction between DRB1*0301and DRB1*0302 in specific heterozygote combinations.

We have thus developed a more informative strategy to deal with DRB;this strategy, which consists of the additional use of non-conserved(Type 2) primers permits the clear elucidation of even the most complexcombination of the four DRB sequences that might be present in anindividual. These non-conserved primers, as opposed to allele-specificprimers, are designed to be used in reactions performed simultaneouslywith the reactions using Type 1 primers and aim at selectivelyamplifying certain ladders from the complex sequencing patterns withoutrequiring previous typing information.

Analysis of the sequence variability of the second exon of the DRB geneshas allowed us to identify two regions which could be used to designnon-conserved (Type 2) primers: 1) codons 5-13; and 2) codons 29-35. Thesequence of the former region follows a group-specific sequence pattern,i.e., a sequence shared by groups of alleles at individual loci. Thelater region exhibits a scattered nucleotide polymorphism in DRB1 andDRB3, DRB4 and DRB5 genes. We designed five different non-conservedprimers annealing to these two polymorphic regions: 1) DRB23 (specificfor DR2-DRB1 ladders); 2) DRB24 (specific for DRw17-, DRw18-, DRw13-,DRw14-, DRw11-, DRw12-, and DRw8- DRB1 ladders); 3) DRB25 (specific forDR4- DRB1 ladders); 4) DRB16 and 5) DRB17, the latter two primersannealing to the second region of moderate polymorphism (from 1 to 5nucleotides different among the known alleles for each locus) (Tables Iand IV). Because of the different nature and distribution of mismatchesbetween these primers and the different DRB templates, the type oftemplates amplified selectively by these primers will be different. EachOf the first three primers will amplify up to two DRB1 cDNAs in anygiven heterozygote and will not amplify any DRB3, DRB4 or DRB5 cDNAs. Onthe contrary, the use of primers DRB16 and DRB17 will allow the randomselective amplification of certain transcripts from DRB1, DRB3, DRB4and/or DRB5 loci in most heterozygote combinations. We therefore testedthese primers in order to determine which combination would give thebest discriminatory results for DRB typing. Furthermore, since thesequences of these primers carry from 0-12 mismatches with the sequencesof the known DRB alleles at the different DRB loci, their use allowed usto determine the number of mismatches between the primers and each ofthe possible cDNAs that are required to obtain such selectiveamplification of DRB transcripts. The specific combinations of primersused for the cDNA/PCR/sequencing reactions are shown in Table II above.The results of this analysis are shown below.

                                      TABLE IV                                    __________________________________________________________________________    Contribution of Nucleotide Base Pair Mismatches Between 5' Amplification      Primers and DRB Alleles to the Selective Amplification of Allelic             And/or Non-Allelic DRB Transcripts                                            Table IIIa. Mismatches between Type 2 DRB-primers and DRB alleles at          different loci.                                                               __________________________________________________________________________    DRB1                                                                                      *1601-2                                                                           *1401-2    DR5x6##                                                *0101-3                                                                           *1501                                                                             *1502                                                                             *0301/1301-2                                                                         *0401-8                                                                           *1101-4                                                                             *0801-3                                                                           *1201                                                                             *0701                                __________________________________________________________________________    DRB16                                                                             0   2   1   4      3   2     3   5   4                                    DRB17                                                                             4   4   5   0      3   2     3   1   3                                    DRB23                                                                             5   0   0   7      4   4     7   8   8                                    DRB24                                                                             6   5   5   0      4   0     0   0   12                                   DRB25                                                                             6   4   4   8      0   8     8   8   5                                    __________________________________________________________________________    DRB1         DRB3/DRB4/DRB5                                                       *0901                                                                             *1001                                                                              DRB5# DRB3*0101                                                                            DRB3*0201                                                                            DRB3*0301                                                                            DRB4                                  __________________________________________________________________________    DRB16                                                                             1   3    3     4      5      4      2                                     DRB17                                                                             5   4    1     0      1      0      2                                     DRB23                                                                             2   4    6     5      5      5      3                                     DRB24                                                                             5   4    6     5      5      5      3                                     DRB25                                                                             5   4    8     6      5      5      4                                     __________________________________________________________________________     #DRB5 gene from cell line AMAI has an additional nucleotide substitution      in the first base of codon 30, in comparison with DRB5 genes of other DR2     haplotypes.                                                                   ##The DRB1 gene of this specificity (DR5x6) has been given this arbitrary     designation according to serological, RFLP and sequence information.     

                  TABLE V                                                         ______________________________________                                        Selective Amplification of DRB and DQB1 cDNAs                                 In Combinations of Alleles Mismatched with                                    Type 2 Oligonucleotides (#)                                                   Haplotypes         Selected Alleles                                                                          DO Primer                                      ______________________________________                                        DRB1*1301,DRB3*0101/                                                                             DRB1*1601   DRB16                                          DRB1*1601,DRB5*0201                                                           DRB1*1301,DRB3*0101/                                                                             DRB1*0801   DRB16                                          DRB1*0801                                                                     DRB1*0301,DRB3*0101/                                                                             DRB1*1601   DRB16                                          DRB1*1601,DRB5*0201                                                           DR5x6,DRB3*0101/DRB1*0801                                                                        DRB1*0801   DRB16                                          DR5x6,DRB3*0101/DRB1*08011                                                                       DRB3*0101   DRB17                                          DR5x6,DRB3*0101/   DR5x6       DRB16                                          DRB1*0301,DRB3*0101                                                           DRB1*1101,DRB3*0201/                                                                             DRB3*0201   DRB16                                          DRB1*1501,DRB5*0101                                                           DRB1*1101,DRB3*0201/                                                                             DRB5*0101   DRB17*                                         DRB1*1501,DRB5*0101                                                           DRB1*1201,DRB3*0201/                                                                             DRB1*1101   DRB16                                          DRB1*1101,DRB3*0201                                                           DRB1*1201,DRB3*0201/                                                                             DRB1*1201 + DRB17                                          DRB1*1101,DRB3*0201                                                                              DRB3*0201                                                  DRB1*0405,DRB4*0101/                                                                             DRB1*0405 + DRB16                                          DRB1*0301,DRB3*0101                                                                              DRB4*0101                                                  DR5x6,DRB3*0101/   DRB1*1101 + DRB16                                          DRB1*1101,DRB3*0201                                                                              DR5x6                                                      DRB1*1501,DRB5*0101                                                                              DRB1*1501   DRB16                                          DRB1*1601,DRB5*0201/                                                                             DRB1*0401 + DRB17                                          DRB1*0401,DRB4*0101                                                                              DRB5*0201                                                  DRB1*1601,DRB5*0201/                                                                             DRB1*1601 + DRB16**                                        DRB1*0401,DRB4*0101                                                                              DRB1*0401                                                  DRB1*1601,DRB5*0201                                                                              DRB1*1601   DRB16                                          DRB1*1601,DRB5*0201                                                                              DRB5*0201   DRB17                                          DRB1*1602,DRB5*0202                                                                              DRB1*1602   DRB16                                          DRB1*0401,DRB4*0101                                                                              DRB4*0101   DRB16                                          DQB1*0604/DQB1*0502                                                                              DQB1*0604   DQB6                                           DQB1*0301/DQB1*0101                                                                              DQB1*0301   DQB6                                           DQB1*0301/DQB1*0101                                                                              DQB1*0501   DQB14                                          DQB1*0201/DQB1*0603                                                                              DQB1*0201   DQB6                                           DQB1*0604/DQB1*0301                                                                              DQB1*0604   DQB15                                          DQB1*0301/DQB1*0502                                                                              DQB1*0301   DQB6                                           DQB1*0603/DQB1*0101                                                                              DQB1*0603   DQB6                                           DQB1*0603/DQB1*0101                                                                              DQB1*0501   DQB14                                          DQB1*0201/DQB1*0101                                                                              DQB1*0501   DQB14                                          DQB1*0201/DQB1*0302                                                                              DQB1*0201/  DQB6***                                                           DQB1*0302                                                  DQB1*0201/DQB1*0502                                                                              DQB1*0201   DQB6                                           ______________________________________                                         (#) In this Table we only show representative examples of haplotypic          combinations lacking those alleles the primers are fully matched with, fo     reasons of simplicity (see Table IV). Whenever these primers were used in     heterozygotes carrying the alleles they specifically recognize, these         alleles were selectively amplified. Note that in the examples shown in th     Table the nonconserved primers selectively amplified the templates closer     in sequence to the primer. The DRB and DQB1 alleles composing these           haplotypes are shown under "haplotypes". The selected alleles and the         primers used are indicated in the two other columns. More than one            individual was tested for some of the heterozygote combinations listed in     this Table.                                                                   *DRB5*0101 and DRB3*0201 templates both have one mismatch with primer         DRBI17. Selection of DRB5*0101 could be related to the differential           positioning of the mismatch with respect to the primer.                       **A Weaker DRB4*0101 template was also observed.                              ***Despite the presence of a mismatch between these two DQB1 alleles,         primer DODQB6 was not able to select either of them.                     

These primers were able to selectively amplify certain DRB templates inall the heterozygous combinations tested. In heterozygotes carrying thealleles these primers are matched with, these alleles were selectivelyamplified; in heterozygotes not carrying the alleles specificallyrecognized by the primers, the DRB templates which had the fewest basepair mismatches with the primers were selectively amplified in the PCR.Specific examples of the latter are shown in Table V. As shown in TableV, Type 2 primers could differentially amplify DRB transcripts from thecombinations of allelic cDNAs that differ from each other in as few asone nucleotide substitution, provided that high stringency annealingconditions are used for the PCR (annealing at 55° C.). For example, inthe heterozygote combination DRw13/DRw8-Dw8.1, the DRB1*0801 allele (3mismatches with the primer) was selected over DRB1*1301 and DRB3*0101genes (each has 4 mismatches with the primer) by the DRB16oligonucleotide primer. Although DRB3*0101 or DRB3*0201 and DRB5*0101genes all harbour one mismatch with primer DRB17, this oligonucleotideselected the DRB5*0101 sequence in a DRw11/DRw15 heterozygote (Table V).It is possible that the differential positioning of the mismatcheswithin the sequence recognized by the oligonucleotide also has aninfluence on the stability of the primer/cDNA complex and hence on theoutcome of the PCR.

The ability of non-conserved primers to select certain alleles inheterozygote combinations was also tested for DQB1 genes (Table V). Aswith DRB-specific Type 2 primers, the use of high temperatures (55° C.)in the annealing step of the PCR was required for achieving theselective amplification of single DQB1 alleles in heterozygotes withnon-conserved primers. For instance, when annealing of primer DQB6 wasallowed to proceed at 37° C. in cDNAs from a DQB1*0301/DQB1*0501 and aDQB1*0201/DQB1*0603 heterozygote, both alleles in both heterozygoteswere equally amplified. At 55° C., the allele with the most homologoussequence to the 5' primer, was amplified over the other in the PCR.Combinations of alleles both differing from the primer in twonucleotides but in different relative position were also differentiallyamplified with a non-conserved primer. For instance, primer DQB6selected the DQB1*0604 sequence in a DQB1*0604/DQB1*0502 heterozygote(Table V). Five nucleotides separate the two mismatches between theDQB1*0604 allele and the DQB6 primer, whereas only two nucleotidesseparate the mismatches between the DQB1*0502 and the primer.

These results clearly indicate that the oligonucleotide primersannealing to polymorphic regions at the 5' end of the target cDNAs canbe tailored to achieve a reproducible selective amplification of alimited number of DRB or DQB templates in complex heterozygouscombinations. Although the use of Type 1 primers allows the unambiguoussequencing of all possible DQA, DQB, DPA and DPB heterozygotes, such anapproach will not give absolute discriminatory information for all DRBheterozygotes. We have shown that the simultaneous use of Type 1 andType 2 primers for DRB will permit the clear elucidation of even themost complex of all DRB heterozygote combinations. When DRB-SBT is usedfor typing purposes, we perform three Type 2-reactions (using DRB23, 24and 25) simultaneously with a Type 1-reaction (Table II). Thesimultaneous use of these reactions using these primers has the highestdiscriminatory power for complete DRB typing in a single run and allowsthe identification of novel sequence heterogeneity. Only one Type 1reaction is required for DQB1, one for DQA1, one for DPA1 and one forDPB1 (Table II).

EXAMPLE III Determining Unknown HLA Type of Subjects by DirectSequencing of the Second Exon of Class II Genes

Routine HLA typing of large populations of individuals for sequencepolymorphisms can be performed by the use of the methodology reportedhere which can also identify previously unknown allelic variants. FIGS.2A and 2B show a flow-chart for the-routine protocol used to determinesequence allelism of individuals of unknown HLA types.

1. Employment of Primer Combinations for cDNA, PCR and Direct SequencingUsing RNA as Initial Template

For synthesizing cDNA molecules, the present invention provides singlestrand DNA anti-sense oligonucleotide primers that anneal to conservedregions of the gene mRNAs to be reverse transcribed, amplified andsequenced. These oligonucleotide primers include an oligonucleotidesequence that: (1) anneals to a conserved region (codons 105 through111) shared by all the alleles at all the DRB loci, the latter beingDRB1, DRB3, DRB4 and DRB5, respectively (e.g., primer DRB20). Foursimultaneous cDNA reactions (one per tube) are performed for DRB typing,all using primer DRB20 (reactions A, B, C and D in Table II and FIG.2A); (2) anneals to a conserved region (codons 105 through 111) sharedby all the alleles at the DQB locus (e.g. primer DQB7) (reaction E inTable II and FIG. 2A); (3) anneals to a conserved region (codons 147through 157) shared by all the alleles at the DQA locus (e.g. primerDQA9) (reaction F in Table II and FIG. 2A); (4) anneals to a conservedregion (codons 105 through 111) shared by all the alleles at the DPBlocus (e.g. primer DPB11) (reaction G in Table II and FIG. 2A); (5)anneals to a conserved region (codons 104 through 110) shared by all thealleles at the DPA locus (e.g. primer DPA14) (reaction H in Table II andFIG. 2A); (6) anneals to a conserved region (codons 222 through 228)shared by all the alleles at the DPA locus (e.g. primer DPA19) (reactionI in Table II and FIG. 2A). The specific oligonucleotides added to eachof these reactions once the cDNA synthesis is done in order to amplifyand sequence the products are indicated below as well as in Table II andin FIG. 2.

To amplify cDNA molecules corresponding to each expressed DRB loci ofeach chromosome (DRB1 and DRB3 or DRB4 or DRB5, depending on thehaplotype--isotypic complexity--), a conserved oligonucleotide primerwhich anneals to codons -32 to -26 (e.g. oligonucleotide DRB11) is addedto one of the four tubes where the cDNA synthesis reactionscorresponding to DRB genes took place. The combination of the cDNAsynthesis reaction primer and the newly added conserved primer is usedto amplify all the alleles at all DRB loci expressed by a givenindividual. Each of the remaining three tubes containing DRB cDNAproducts receives one of three different non-conserved oligonucleotides(also called Type 2) annealing to codons 7-13 (e.g. primer DRB23), 5-11(e.g. primer DRB24), 6-13 (e.g. primer DRB25), respectively. Eachnon-conserved primer is designed to favor the amplification of cDNAscorresponding to different groups of alleles at the DRB1 locus.Comparison of the sequencing ladders generated by these four reactionsallows complete and accurate interpretation of the sequencescorresponding to each of the four possible DRB genes expressed by agiven individual (one or two for each of the parental chromosomes).

For the DQB1 locus, a conserved oligonucleotide primer which anneals tocodons 1-7 of the DQB cDNAs (e.g. primer DQB13) can be used foramplifying each of the DQB1 genes expressed in any given individual (onefor each parental chromosome). In the case of the DQA1 locus, aconserved single strand DNA oligonucleotide primer useful for amplifyingeach of the DQA1 genes expressed in any given subject anneals to codons-10 to -4 of the DQA1 cDNA (e.g. primer DQA10). For the DPB1 locus, aconserved oligonucleotide (e.g. primer DPB10) annealing to codons -19 to-13, is used to amplify each of the expressed DPB1 genes in any givensubject. For DPA1 locus, a conserved oligonucleotide (e.g. primer DPA15)annealing to codons -23 to -17, is used to amplify each of the expressedDPA1 genes in a given subject. In a separate reaction, conserved primerDPA18, annealing to codons 59-65 of the DPA1 cDNAs is used incombination with the cDNA primer DPA19 to amplify each of the expressedDPA1 genes in any individual. This second DPA1 reaction is targeted at asecond polymorphic region of this gene.

Primers useful in direct sequencing the polymerase chain reactionproducts corresponding to DRB loci include an anti-sense oligonucleotideprimer (e.g. DRB12) annealing to codons 87-94 of all the alleles at DRBloci; this primer is used for sequencing the products generated by thefirst of the four DRB reactions. For direct sequencing the polymerasechain reaction products generated with the other three DRB reactions, ananti-sense oligonucleotide annealing to codons 97-103 of all the allelesat DRB1 locus can be used (e.g. primer DRB30). The use of a differentsequencing oligonucleotide in these three DRB reactions allows readingof downstream polymorphic regions of DRB1 genes not seen in the firstDRB reaction which uses the example sequencing primer DRB12. Primersuseful in direct sequencing the polymerase chain reaction productscorresponding to DQB1 locus include an anti-sense oligonucleotide primer(e.g. DQB5) annealing to codons 78-83 of all the alleles at this locus.Direct sequencing of polymerase chain reaction products corresponding toDQA1 locus include an anti-sense oligonucleotide primer (e.g. DQA29)annealing to codons 88-95 of all the alleles at this locus. Directsequencing of polymerase chain reaction products corresponding to DPB1locus include a sense oligonucleotide primer (e.g. DPB13) annealing tocodons 12/-5 of all the alleles at this locus. For direct sequencing ofpolymerase chain reaction products for the DPA1 reaction which usedprimers DPA14 and DPA15, an anti-sense oligonucleotide annealing tocodons 88-94 of all the alleles at this locus can be used (e.g. primerDPA16). For direct sequencing of polymerase chain reaction products forthe DPA1 reaction which used primers DPA19 and DPA18, an anti-senseoligonucleotide annealing to codons 214-220 of all the alleles at thislocus can be used (e.g. primer DPA20).

2. Employment of Primer Combinations for PCR and Direct Sequencing UsingDNA Templates

To amplify DNA molecules corresponding to each DRB loci of eachchromosome a conserved anti-sense oligonucleotide primer annealing tobase pairs 18-38 of intron 3 (e.g. oligonucleotide DRB1406) is added toeach of four PCR reaction tubes (reactions S, V, T and U in Table II andFIG. 2B). Each of these four tubes will receive a different additionaloligonucleotide annealing to codons -4 to +3 (e.g. primer DRB22), tocodons 7-13 (e.g. primer DRB23), 5-11 (e.g. primer DRB24), 6-13 (e.g.primer DRB25), respectively. The first reaction is used to amplify allthe alleles at all DRB loci carried by a given individual. Each of theremaining three reactions is designed to favor the amplification of DNAcorresponding to different groups of alleles at the DRB1 locus. As withRNA templates, comparison of the sequencing ladders generated by thesefour reactions allows complete and accurate interpretation of thesequences corresponding to each of the four possible DRB genes expressedby a given individual (one or two for each of the parental chromosomes).

For the DQB1 locus, two conserved oligonucleotide primers which annealto codons 88-94 (e.g. primer DQB932) and 11-17 (e.g. primer DQB931) or1-7 (e.g. primer DQB13) can be used for amplifying each of the DQB1genes carried by any given individual (one for each parental chromosome)(reaction W in Table II and FIG. 2B). For the DPB1 locus, two conservedoligonucleotides (a primer, e.g. DPB14, annealing to base pairs -42 to-62 of intron 2, and a primer e.g. DPB15, annealing base pairs 39-59 ofintron 3) are used to amplify each of the DPB1 genes carried by anygiven subject (reaction X in Table II and FIG. 2B). For DPA1 locus aconserved oligonucleotide such as DPA10 (annealing to base pairs -69 to-50 of intron 2) and DPA11 (annealing to base pairs 55-71 of intron 3)are used to amplify each of the DPA1 genes carried by a given subject(reaction Y in Table II and FIG. 2B).

Primers useful in direct sequencing the polymerase chain reactionproducts generated from DNA templates corresponding to DRB loci includean anti-sense oligonucleotide primer (e.g. DRB12) annealing to codons87-94 of all alleles at DRB loci; this primer is used for sequencing theproducts generated by the first of the four DRB reactions. For directsequencing the polymerase chain reaction products generated with theother three DRB reactions, a sense oligonucleotide annealing to codons39-46 of all the alleles at DRB1 locus can be used (e.g. primerDRB1400). The use of a different sequencing oligonucleotide in thesethree DRB reactions allows reading of downstream polymorphic regions ofDRB1 genes not seen in the first DRB reaction which uses the examplesequencing primer DRB12. Primers useful in direct sequencing thepolymerase chain reaction products corresponding to DQB1 locus includean anti-sense oligonucleotide primer (e.g. DQB5) annealing to codons78-83 of all the alleles at this locus. Direct sequencing of polymerasechain reaction products corresponding to DPB1 locus include ananti-sense oligonucleotide primer (e.g. DPB16) annealing to base pairs1-21 of intron 3 of all the alleles at this locus. For direct sequencingof polymerase chain reaction products for the DPA1 reaction ananti-sense oligonucleotide annealing to codons 76-82 of all the allelesat this locus can be used (e.g. primer DPA12).

Procedure for Determining Unknown HLA Type

A subject of unknown HLA type, diseased or not, is to be typed for ClassII HLA polymorphism. From 10 to 50 mL of peripheral blood are drawn. Theperipheral blood mononuclear cells are prepared by centrifugation overFicoll-Hypaque gradients. The cells are then lysed in guanidiumisothyocianate and total cellular RNA prepared using conventionalmethods (either by centrifugation on cesium chloride gradients, whichlasts about 16 hours, or by the guanidiumisothyocianate-phenol-chlorophorm extraction method, which can beperformed in less than 4 hours. See Gouuh, supra (1988); Johns et al.,Anal. Biochem., 180:276 (1989). Otherwise genomic DNA from these cellsor other sources (hair, blood stains, sperm, etc.) can be prepared withconventional methods such as provided by Higuchi, R. in PCR Technology,Erlich, M. (ed.), Stockton Press:31 (1989). DQB1, DQA1, DRB (DRB1,DRB3/4/5), DPA1 and DPB1 cDNA molecules are synthesized from total RNAusing locus-specific primers. Approximately, one microgram of RNA isreverse transcribed with MoLVRT (reverse transcriptase) and DRB(CODRB20), DQB (CODQB7), DQA (CODQA9), DPB (DPB11) and DPA (DPA14,DPA19) (optional)--specific non-sense primers in a 20 uL final volumereaction (30-60 minute incubation). The reaction for each Class II geneis performed in a different tube, but they can be performed in the sametube if preferred. For routine purposes, four simultaneous reactions areperformed for DRB, one for DQB, one for DQA1, one for DPB1, and two forDPA1 gene products.

Once these reactions are completed, the enzymatic amplification of therespective cDNA molecules is then performed by directly adding to the 20uL reverse transcription reaction, the reagents needed for theamplification step. Alternatively, if DNA is used, the primercombinations used for the PCR are those shown in Table II herein (theanti-sense primers as well as the sense primers will be different). Thisincludes the PCR reagents and appropriate conserved and non-conservedoligonucleotide primers. This example uses four reactions for DRB (tubes1, 2, 3 and 4), one for DQB (tube 5), one for DQA (tube 6), one for DPB(tube 7), and two for DPA (tubes 8 and 9, respectively). Reactions 2, 3and 4 incorporate primers DRB23, DRB24 and DRB25, respectively. Forrapid typing (in less than 24 hours), the latter are the preferredcombinations. Alternative combinations of the primers that can be usedare shown in Table II.

Once completed, the reactions are spun-dialyzed for about 15 minutesusing Centricon (Amicon, Ultrafree (milli-pore)) or similar columns toremove unincorporated primers and dNTPs. The retentate or one half ofthe recovered retentate for each reaction is then directly sequencedusing Taq polymerase and the primers described in Table II for eachcombination of primers used in the cDNA/PCR reactions using P-32end-labeled (10 minutes) locus-specific sequencing primers (35 minutes).

The sequencing reactions products are loaded on an acrylmide gel,electrophoresed in 2-3 hours and exposed to X-ray films for 4-12 hours.The gels are read and results from gels are compared to nucleotidesequences corresponding to all possible alleles.

Comparisons can be made visually using the naked eye or using a personalcomputer and a software package including the nucleotide sequences ofall alleles of all haplotypes and routines which indicate how thecomparison is to be performed as well as subroutines which will allowidentification of new allelic sequences.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 49                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB7                                     (B) LOCATION: Anneals to codons 105 to 111 of the                             DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGTGGTTGAGGGCCTCTGTCC21                                                       (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB20                                    (B) LOCATION: Anneals to codons 105 to 111 of the                             DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GTGCTGCAGGGGCTGGGTCTT21                                                       (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQA9                                     (B) LOCATION: Anneals to codons 148 to 155 of the                             DQA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GGTGAGGTTACTGATCTTGAAG22                                                      (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB13                                    (B) LOCATION: Anneals to codons 1 to 7 of the                                 DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      AGAGACTCTCCCGAGGATTTC21                                                       ArgAspSerProGluAspPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DBR22                                    (B) LOCATION: Anneals to codons -4 to +3 of the                               DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      CTGGCTTTGGCTGGGGACACC21                                                       LeuAlaLeuAlaGlyAspThr                                                         11                                                                            (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB11                                    (B) LOCATION: Anneals to codons -33 to -26 of the                             DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      TGTTCTCCAGCATGGTGTGTC21                                                       PheSerSerMetValCysLeu                                                         30                                                                            (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQA10                                    (B) LOCATION: Anneals to codons -10 to -4 of the                              DQA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      CTGTCCTCCGTGATGAGCCC20                                                        LeuThrThrValMetSerPro                                                         10-5                                                                          (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB932                                   (B) LOCATION: Anneals to codons 88 to 94 of the                               DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      TCGCCTCTGCAGGGTCGCGCG21                                                       (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB931                                   (B) LOCATION: Anneals to codons 11 to 17 of the                               DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      TTTAAGGGCATGTGCTACTTC21                                                       PheLysGlyMetCysTyrPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB30                                    (B) LOCATION: Anneals to codons 97 to 104 of the                              DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     ATGGGGAGATGGTCACTGTGG21                                                       (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB30                                    (B) LOCATION: Anneals to codons 97 to 103 of the                              DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     AGGATACACAGTCACCTTAGG21                                                       (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB5                                     (B) LOCATION: Anneals to codons 78 to 83 of the                               DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GTAGTTGTGTCTGCACAC18                                                          (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB12                                    (B) LOCATION: Anneals to codons 87 to 94 of the                               DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     GCCGCTGCACTGTGAAGCTC20                                                        (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQA29                                    (B) LOCATION: Anneals to codons 82 to 89 of the                               DQA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     CACGGTTCCGGTAGCAGCGGTAG23                                                     (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQA30                                    (B) LOCATION: Anneals to codons 19 to 24 of the                               DQA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     TACGGTCCCTCTGGCCAG18                                                          TyrGlyProSerGluGln                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB1400                                  (B) LOCATION: Anneals to codons 38 to 45 of the                               DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     GCGCTTCGACAGCGACGTGG20                                                        ValArgPheAspSerAspValGly                                                      4045                                                                          (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB1401                                  (B) LOCATION: Anneals to codons 98 to 104 of the                              DRB1*0701-2 transcript of HLA class II                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     GAGGTGACTGTGTATCCTGAC21                                                       GluValThrValTyrProAsp                                                         100                                                                           (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB1402                                  (B) LOCATION: Anneals to codons 142 to 148 of the                             DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     GATCAGGCCTGTGGACACCAC21                                                       (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB1403                                  (B) LOCATION: Anneals to codons 127 to 133 of the                             DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     CCGGAACCACCTGACTTCAAT21                                                       (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB1406                                  (B) LOCATION: Anneals to bp18-38 to intron 33 of                              the DRB1, DRB3, DRB4 and DRB5 transcripts of                                  HLA class II                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     GCCAAGAGTGGGCCTCGCAGC21                                                       (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB825                                   (B) LOCATION: Anneals to codons 79 to 85 of the                               DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     AACCCCGTAGTTGTGTCTGCA21                                                       (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB824                                   (B) LOCATION: Anneals to codons 1 to 7 of the                                 DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     GGGGACACCCGACCACGTTTC21                                                       GlyAlaThrArgProArgPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB10                                    (B) LOCATION: Anneals to codons -19 to -13 of the                             DPB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     CGGACAGTGGCTCTGACGGCG21                                                       ArgThrValAlaLeuTyrAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB11                                    (B) LOCATION: Anneals to codons 105 to 111 of the                             DPB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     GTTGTGGTGCTGCAAGGGCCC21                                                       (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB12                                    (B) LOCATION: Anneals to codons 97 to 103 of the                              DPB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     CTTGGAGGGGGAAACATTCAC21                                                       (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB13                                    (B) LOCATION: Anneals to codons -5 to +2 of the                               DPB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     TACTGATGGTGCTGCTCACAT21                                                       LeuLeuMetValLeuLeuThrSer                                                      12-5                                                                          (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB14                                    (B) LOCATION: Anneals to bp-42/-46 to intron 2 of                             the DPB1 transcript of HLA class II                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     AGAGGGAGAAAGAGGATTAGA21                                                       (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB15                                    (B) LOCATION: Anneals to bp39-59 to intron 3 of                               the DPB1 transcript of HLA class II                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     GCCCTGGGCACGGGCCCGCGG21                                                       (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB16                                    (B) LOCATION: Anneals to bp1-21 to intron 3 of                                the DPB1 transcript of HLA class II                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     CGGCCCAAAGCCCTCACTCAC21                                                       (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPB17                                    (B) LOCATION: Anneals to bp-6/-26 to intron 2 of                              the DPB1 transcript of HLA class II                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     CGCTCATGTCCGCCCCCTCCC21                                                       (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA14                                    (B) LOCATION: Anneals to codons 104 to 110 of the                             DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     GTCAATGTGGCAGATGAGGGT21                                                       (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA15                                    (B) LOCATION: Anneals to codons -17 to -23 of the                             DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     CATATCAGAGCTGTGATCTTG21                                                       (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA16                                    (B) LOCATION: Anneals to codons 88 to 94 of the                               DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     CTTGGGAAACACGGTCACCTC21                                                       (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA17                                    (B) LOCATION: Anneals to codons -3 to -9 of the                               DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     CTGCTGAGTCTCCGAGGAGCT21                                                       LeuLeuSerLeuArgGlyAla                                                         (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA10                                    (B) LOCATION: Anneals to bp-69/-50 of intron 2 of                             the DPA1 transcript of HLA class II                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     CTCTAGCTTTGACCACTTGC20                                                        (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA11                                    (B) LOCATION: Anneals to bp55-71 to intron 3 of                               the DPA1 transcript of HLA class II                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     AGTCTGAGGGTGGCAGAGAGG21                                                       (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA12                                    (B) LOCATION: Anneals to codons 76 to 82 of the                               DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     GGCCTGAGTGTGGTTGGAACG21                                                       (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA18                                    (B) LOCATION: Anneals to codons 59 to 65 of the                               DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     CTGGCTAACATTGCTATATTG21                                                       LeuAlaAsnIleAlaIleLeu                                                         6065                                                                          (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA19                                    (B) LOCATION: Anneals to codons 222 to 228 of the                             DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     GGTCCCCTGGGCCCGGGGGTC21                                                       (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA20                                    (B) LOCATION: Anneals to codons 214 to 220 of the                             DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     GCCAGAACGCAGAGACTTTAT21                                                       (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DPA21                                    (B) LOCATION: Anneals to codons 68 to 74 of the                               DPA1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     AACTTGAATACCTTGATCCAG21                                                       AsnLeuAsnThrLeuIleGln                                                         70                                                                            (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB23                                    (B) LOCATION: Anneals to codons 7 to 13 of the                                DRB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     TTCTTGCAGCAGGATAAGTA20                                                        PheLeuGlnGlnAspLysTyr                                                         10                                                                            (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB24                                    (B) LOCATION: Anneals to codons 5 to 11 of the                                DRB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     CCACGTTTCTTGGAGTACTCT21                                                       ProArgPheLeuGlyTyrSer                                                         510                                                                           (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB25                                    (B) LOCATION: Anneals to codons 6 to 13 of the                                DRB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     TTTCTTGGAGCAGGTTAAACA21                                                       ArgPheLeuGluGlnValLysHis                                                      10                                                                            (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB16                                    (B) LOCATION: Anneals to codons 29 to 35 of the                               DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     AGATGCATCTATAACCAAGAG21                                                       ArgCysIleTyrAsnGlnGlu                                                         3035                                                                          (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DRB17                                    (B) LOCATION: Anneals to codons 29 to 35 of the                               DRB1, DRB3, DRB4 and DRB5 transcripts of HLA                                  class II                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     AGATACTTCCATAACCAGGAG21                                                       ArgTyrPheHisAsnGlnGlu                                                         3035                                                                          (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB6                                     (B) LOCATION: Anneals to codons -8 to -2 of the                               DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     CTGAGCACCCCAGTGGCTGAG21                                                       LeuSerThrProValAlaGlu                                                         5                                                                             (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB14                                    (B) LOCATION: Anneals to codons -8 to -2 of the                               DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     CTGAGCTCCTCACTGGCTGAG21                                                       LeuSerSerSerLeuAlaGlu                                                         5                                                                             (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer DQB15                                    (B) LOCATION: Anneals to codons -8 to -2 of the                               DQB1 transcript of HLA class II                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     CTGAGCACCTCGGTGGCTGAG21                                                       LeuSerThrSerValAlaGlu                                                         5                                                                             __________________________________________________________________________

Applicants state that the paper copy of the above "Sequence Listing"Section of the present application, and the computer readable form ofthe same submitted therewith, are the same.

What is claimed:
 1. A method for determining the genotype of one or moreHLA Class H polymorphic gene loci of interest selected from the groupconsisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and --DPA1, of nsubject in a sample containing subject RNA using a combination ofconserved and non-conserved oligonucleotide primers, said methodcomprising:(a) synthesizing in separate reactions, from RNA isolatedfrom said sample, a cDNA molecule for each allele of each gene locus ofinterest, using a conserved oligonucleotide antisense primer thatanneals to a nucleotide region conserved between each allele of eachgene loci to be genotyped; (b) amplifying said cDNA by at least oneamplifying reaction to generate sufficient first and second doublestranded amplifying reaction products for sequencing, using in aseparate first amplifying reaction for each of said gene loci ofinterest encoded by the 3'-end of the alpha 1 domain exon, anoligonucleotide primer pair comprising a first conserved primer and asecond conserved primer or, when said gene locus is HLA-DQB or HLA-DRB,optionally a first nonconserved primer for each of said gene loci ofinterest, wherein one of said first and second conserved oligonucleotideprimers anneals to at least one allele of each of said loci of interest;(c) sequencing directly at least one strand of said amplifying reactionproduct of each of said gene loci of interest to produce at least onesequencing ladder for each of said gene loci of interest, using in eachsequencing reaction a third conserved oligonucleotide primer for each ofsaid gene loci of interest; (d) comparing the nucleotide sequenceobtained from each sequencing ladder to known sequences for each suchgene locus; and, optionally when said gene locus is HLA-DQB or HLA-DRB,(e) comparing the nucleotide sequence of each sequencing ladderoptionally obtained with a conserved oligonucleotide primer pair to thenucleotide sequence of each allele of such gene locus amplified with aconserved/nonconserved oligonucleotide primer pair, wherein said geneloci of interest are one or more gene loci whereby the Zenotype of saidHLA Class II gene locus is obtained, wherein when said gene locus isHLA-DQA1 (a) said conserved oligonucleotide antisense primer is depictedin SEQ. ID. NO: 3; (b) said first and second conserved primers aredepicted in SEQ. ID. NO: 3 and SEQ. ID. NO: 7: and (c) said thirdconserved primer is depicted in SEQ. ID. NO: 14 or SEQ. ID. NO: 15;wherein when said gene locus is HLA:DOB1 said combination of conservedand non-conserved oligonucleotide primers is selected from the groupconsisting of (1) (a) said conserved oligonucleotide antisense primer isdepicted in SEQ. ID. NO: 1: (b) said first and second conserved primersare depicted in SEQ. ID. NO: 1 and SEQ. ID. NO: 4; and (c) said thirdconserved primer is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12; (2)(a) said conserved oligonucleotide antisense primer is depicted in SEQ.ID. NO: 1: (b) said tint conserved primer is depicted in SEQ. ID. NO: 1and said first nonconserved primer is depicted SEQ. ID. NO: 47 andwherein annealing of said conserved/nonconserved primer pair occurs atabout 37° C.; and (c) said third conserved primer is depicted in SEQ.ID. NO: 4; (3) (a) said conserved oligonucleotide antisense primer isdepicted in SEQ. ID. NO: 1: (b) said first conserved primer is depictedin SEQ. ID. NO: 1 and said first nonconserved primer is depicted SEQ.ID. NO: 47 and wherein annealing of said conserved/nonconserved primerpair occurs at about 55° C.-60° C.; and (c) said third conserved primeris depicted in SEQ. ID. NO: 10or SEQ. ID. NO: 12; (4) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 1: (b) saidfirst conserved primer is depicted in SEQ. ID. NO: 1 and said firstnonconserved primer is depicted SEQ. ID. NO: 48; and (c) said thirdconserved primer is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12; and(5) (a) said conserved oligonucleotide antisense primer is depicted inSEQ. ID. NO: 1: (b) said first conserved primer is depicted in SEQ. ID.NO: 1 and said first nonconserved primer is depicted SEQ. ID. NO: 49:and (c) said third conserved primer is depicted in SEQ. ID. NO: 10 orSEQ. ID. NO: 12; wherein when said gene locus is HLA-DPB1, saidcombination of conserved and non-conserved oligonucleotide primers isselected from the group consisting of (1) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 24; (b)said first and second conserved primers are depicted in SEQ. ID. NO: 24and SEQ. ID. NO: 23: and (c) said third conserved primer is depicted inSEQ. ID. NO: 25 or SEQ. ID. NO: 26: and (2) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 25; (b)said first and second conserved primers are depicted in SEQ. ID. NO: 25and SEQ. ID. NO: 23: and (c) said third conserved primer is depicted inSEQ. ID. NO: 26; wherein when said gene locus is HLA-DPA1, saidcombination of conserved and non-conserved oligonucleotide primers isselected from the group consisting of (1) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 31; (b)said first and second conserved primers are depicted in SEQ. ID. NO: 31and SEQ. ID. NO: 32; and (c) said third conserved primer is depicted inSEQ. ID. NO: 33 or SEQ. ID. NO: 34; (2) (a) said conservedoligonucleotide and sense primer is depicted in SEQ. ID. NO: 39; (b)said first and second conserved primers are depicted in SEQ. ID. NO: 39and SEQ. ID. NO: 38: and (c) said third conserved primer is depicted inSEQ. ID. NO: 40 or SEQ. ID. NO: 41; (3) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 33; (b)said first and second conserved primers are depicted in SEQ. ID. NO: 33and SEQ. ID. NO: 32; and (c) said third conserved primer is depicted inSEQ. ID. NO: 34; wherein when said gene locus is HLA-DRB, saidcombination of conserved and non-conserved oligonucleotide primers isselected from the group consisting of (1) (a) said conservedoligonucleotide and sense primer is depicted in SEQ. ID. NO: 2: (b) saidfirst and second conserved primers are depicted in SEQ. ID. NO: 2 andSEQ. ID. NO: 6: and (c) said third conserved primer is depicted in SEQ.ID. NO: 11 or SEQ. ID. NO: 13; (2) (a) said conserved oligonucleotideantisense primer is depicted in SEQ. ID. NO: 2; (b) said first conservedprimer is depicted in SEQ. ID. NO: 2 and said first nonconserved primeris depicted in SEQ. ID, NO: 42 and (c) said third conserved primer isdepicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; (3) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) saidfirst conserved primer is depicted in SEQ. ID. NO: 2 and said firstnonconserved primer is depicted in SEQ, ID. NO: 43; and (c) said thirdconserved primer is depicted in SEQ. ID. NO: 11 or SEQ, ID. NO: 13; (4)(a) said conserved oligonucleotide antisense primer is depicted in SEQ.ID. NO: 2; (b) said first conserved primer is depicted in SEQ, ID. NO: 2and said first nonconserved primer is depicted in SEQ. ID, NO: 44: and(c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ.ID. NO: 13; (5) (a) said conserved oligonucleotide antisense primer isdepicted in SEQ. ID. NO: 2; (b) said amplifying has four discreteamplifying steps, and wherein in first discrete amplifying step saidfirst conserved primer is depicted in SEQ. ID. NO: 2 and said secondconserved primer is depicted in SEQ. ID. NO: 6: and in a second discreteamplifying step said first conserved primer is depicted in SEQ. ID. NO:2 and said first nonconserved primer is depicted in SEQ. ID. NO: 42),and in a third discrete amplifying step said first conserved primer isdepicted in SEQ. ID. NO: 2 and said second nonconserved primer isdepicted in SEQ. ID. NO: 43 and in a fourth discrete amplifying stepsaid first conserved primer is depicted in SEQ. ID. NO: 2 and said thirdnonconserved primer is depicted in SEQ. ID. NO: 44; and (c) said thirdconserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13 orSEQ. ID. NO: 5; (6) (a) said conserved oligonucleotide antisense primeris depicted in SEQ. ID. NO: 2; (b) said first conserved primer isdepicted in SEQ. ID. NO: 2 and said first nonconserved primer isdepicted in SEQ. ID. NO: 45; and (c) said third conserved primer isdepicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; (7) (a) said conservedoligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) saidfirst conserved primer is depicted in SEQ. ID. NO: 2 and said firstnonconserved primer is depicted in SEQ. ID. NO: 46: and (c) said thirdconserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; and(8) (a) said conserved oligonucleotide antisense primer is depicted inSEQ. ID. NO: 2; (b) said first and second conserved primers are depictedin SEQ. ID. NO: 2 and SEQ. ID. NO: 5; and (c) said third conservedprimer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO:
 13. 2. A methodfor determining the genotype of one of more HLA Class II polymorphicgene loci of interest selected from the group consisting ofHLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and DPA1 of a subject in a samplecontaining subject genomic DNA using a combination of conserved andnonconserved oligonucleotide primers, said method comprising;(a)amplifying said genomic DNA by at least one amplifying reaction togenerate sufficient tint and second double stranded amplifying reactionproducts for sequencing, using in a separate first amplifying reactionfor each of said gene loci of interest encoded by the 3'-end of thealpha 1 domain exon, an oligonucleotide primer pair comprising a firstconserved primer and either a second conserved primer or a firstnonconserved primer for each of said gene loci of interest, wherein saidconserved primers anneal to a nucleotide sequence conserved between eachallele of each gene loci to be genotyped, and wherein said nonconservedprimer anneals to a nucleotide sequence of a group of two or lessalleles of each gene loci to be genotyped; and (b) sequencing directlyat least one strand of said amplifying reaction product for each of saidgene loci of interest to produce at least one sequencing ladder for eachof said gene loci of interest, using in each sequencing reaction a thirdconserved oligonucleotide primer for each of raid gene loci of interestwhereby a nucleotide sequence is obtained for each sequencing ladder;(c) comparing said nucleotide sequence obtained from each sequencingladder to known sequences for each gene locus of interest; and (d) whena first non-conserved primer is used, comparing said nucleotide sequenceof each sequencing ladder obtained with a conserved oligonucleotideprimer pair to the nucleotide sequence of each allele of such gene locusamplified with a conserved/nonconserved oligonucleotide primer pair,whereby the genotype of one or more HLA Class II gene loci is obtained,wherein when said gene locus is HLA-DQB-1 (a) said first and secondconserved primers are depicted in SEQ. ID. NO: 8and SEQ. ID. NO: 9; and(b) said third conserved primer is depicted in SEQ. ID. NO: 12; whereinwhen said gene locus is HLA-DPB1, said combination of conserved andnon-conserved primers are selected from the group consisting of (1) (a)said first and second conserved primers are depicted in SEQ. ID. NO: 27and SEQ. ID. NO: 28; and (b) said third conserved primer is depicted inSEQ. ID. NO: 29 or SEQ. ID. NO: 30; and (2) (a) said first and secondconserved primers are depicted in SEQ. ID. NO: 27 and SEQ. ID. NO: 29;and (b) said third conserved primer is depicted n SEQ. ID. NO: 30;wherein when said one locus is HLA-DPA1, (a) said first and secondconserved primers are depicted in SEQ. ID. NO: 35 and SEQ. ID. NO: 36;and (b) said third conserved primer is depicted in SEQ. ID. NO: 37;wherein when said gene locus is HLA-DRB, said combination of conservedand non-conserved primers are selected from the group consisting of (1)(a) said first and second conserved primers are depicted in SEQ. ID. NO:20 and said first nonconserved primer is depicted in SEQ. ID. NO: 5; and(b) said third conserved primer is depicted in SEQ. ID. NO: 13 or SEQ.ID. NO: 16; (2) (a) said first conserved primer is depicted in SEQ. ID.NO: 20 and said first nonconserved primer is depicted in SEQ. ID. 43;and (b) said third conserved primer is depicted in SEQ. ID. NO: 13 orSEQ. ID. NO: 16; (3) (a) said first conserved primer is depicted in SEQ.ID. NO: 20 and said first nonconserved primer is depicted in SEQ. ID.NO: 44; and (b) said third conserved primer is depicted in SEQ. ID. NO:13 or SEQ. ID. NO: 16; (4) (a) said first conserved primer is depictedin SEQ. ID. NO: 20 and said first nonconserved primer is depicted inSEQ. ID. NO: 42; and (b) said third conserved primer is depicted in SEQ.ID. NO: 13 or SEQ. ID. NO: 16; (5) (a) said amplifying has four discreteamplifying steps and wherein in a first discrete amplifying step saidfirst conserved primer is depicted in SEQ. ID. NO: 20 and said secondconserved primer is depicted in SEQ. ID. NO: 5, and in a second discreteamplifying step said first conserved primer is depicted in SEQ. ID. NO:20 and said first nonconserved primer is depicted in SEQ. ID. NO: 43,and in a third discrete amplifying step said first conserved primer isdepicted in SEQ. ID. NO: 20 and said second nonconserved primer isdepicted in SEQ. ED. NO: 44 and in a fourth discrete amplifying stepsaid first conserved primer is depicted in SEQ. ID. NO: 20 and saidthird nonconserved primer is depicted in SEQ. ID. NO: 42; and (b) saidthird conserved primer is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO:16; (6) (a) said first and second conserved primers are depicted in SEQ.ID. NO: 13 and SEQ. ID. NO: 22; and (b) said third conserved primer isdepicted in SEQ. ID. NO: 21; (7) (a) said first conserved prime isdepicted in SEQ. ID. NO: 17 and said nonconserved primer is depicted inSEQ. ID. NO: 18; and (b) said third conserved primer is depicted in SEQ.ID. NO: 19; (8) (a) said first conserved primer is depicted in SEQ. ID.NO: 20 and said nonconserved primer is depicted in SEQ. ID. NO: 45; and(b) said third conserved primer is depicted in SEQ. ID. NO: 21 or SEQ.ID. NO: 13; and (9) (a) said first conserved primer is depicted in SEQ.ID. NO: 20 and said nonconserved primer is depicted in SEQ. ID. NO: 46;and (b) said third conserved primer is depicted in SEQ. ID. NO: 21 orSEQ. ID. NO:
 13. 3. A method for determining the genotype of one or moreHLA Class II polymorphic gene loci of interest selected from the groupconsisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and DPA1, of asubject in a sample containing subject RNA using a combination ofconserved and nonconserved oligonucleotide primers, said methodcomprising:(a) synthesizing in separate reactions, from RNA isolatedfrom said sample, a cDNA molecule for each allele of each gene locus ofinterest, using a conserved oligonucleotide antisense primer for each ofsaid gene loci of interest; (b) amplifying said cDNA by at least oneamplifying reaction to generate sufficient first and second doublestranded amplifying reaction products for sequencing, using in aseparate first amplifying reaction of each of said gene loci of interestencoded by the 3'-end of the alpha 1 domain exon, an oligonucleotideprimer pair comprising a first conserved primer and either a secondconserved primer or a first nonconserved primer for each of said geneloci of interest, wherein one of said first and second conservedoligonucleotide primers anneals to at least one allele of each of saidloci of interest; (c) sequencing directly at least one strand of saidamplifying reaction product for each of said gene loci of interest toproduce at least one sequencing ladder for each of said gene loci ofinterest, using in each sequencing reaction a third conservedoligonucleotide primer for each of said gene loci of interest; (d)comparing the nucleotide sequences obtained from each sequencing ladderto known sequences for each such gene locus; (e) when a firstnon-conserved primer is used, comparing the nucleotide sequence of eachsequencing ladder obtained with a conserved oligonucleotide primer pairto the nucleotide sequence of each allele of such gene locus amplifiedwith a conserved/nonconserved oligonucleotide primer pair, wherein saidgene loci of interest are one or more gene loci whereby the genotype ofsaid HLA Class II gene locus is obtained: (f) wherein:i) said conservedoligonucleotide antisense palmer of HLA-DQA1 is depicted in SEQ. ID. No:3, ii) said first and second conserved primer of HLA-DQA1 are depictedin SEQ. ID. NO: 3 and SEQ. ID. NO: 7, and iii) said third conservedprimer of HLA-DQ1 is depicted in SEQ. ID. NO: 14 or SEQ. ID. NO: 15, (g)wherein:i) said conserved oligonucleotide antisense primer of HLA-DQB1is depicted in SEQ. ID. NO: 1, ii) said first and second conservedprimers of HLA-DQB1 are depicted in SEQ. ID. NO: 1 and SEQ. ID. NO: 4,and iii) said third conserved primer of HLA-DBQ1 is depicted in SEQ. ID.NO. 10 or SEQ. ID. NO: 12; (h) wherein:i) said conserved oligonucleotideantisense primer of HLA-DPB1 is depicted in SEQ. ID. NO: 24, ii) saidfirst and second conserved primers of HLA-DPB1 are depicted in SEQ. ID.NO: 24 and SEQ. ID. NO: 23, and iii) said third conserved primer ofHLA-DPB1 is depicted in SEQ. ID. NO: 25 or SEQ. ID. NO: 26; and (i)wherein:i) said conserved oligonucleotide antisense primer of HLA-DRB isdepicted in SEQ. ID. NO: 2, ii) said first and second conserved primersof HLA-DRB are depicted in SEQ. ID. NO: 2 and SEQ. ID. NO: 6, and iii)said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 orSEQ. ID. NO: 13 or SEQ. ID. NO:
 5. 4. The method according to claim 3wherein:(a) said conserved oligonucleotide antisense primer of HLA-DQB1is depicted in SEQ. ID. NO: 1; (b) said first conserved primer ofHLA-DQB1 is depicted in SEQ. ID. NO: 1 and said first nonconservedprimer of HLA-DQB1 is depicted in SEQ. ID. NO: 47 and wherein annealingof said conserved/nonconserved primer pair occurs at about 37° C.; and(c) said third conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO:4.
 5. The method according to claim 3 wherein:(a) said conservedoligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO:1; (b) said first conserved primer of HLA-DQB1 is depicted in SEQ. ID.NO: 1 and said first nonconserved primer of HLA-DQB1 is depicted in SEQ.ID. NO: 46 and wherein annealing of said conserved/nonconserved primerpair occurs at about 55° C.-60° C.; and (c) said third conserved primerof [HLA-DQB!] HLA-DOB1 is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO:12.
 6. The method according to claim 3 wherein:(a) said conservedoligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO:1; (b) said first conserved primer of HLA-DQB1 is depicted in SEQ. ID.NO: 1 and said first nonconserved primer of HLA-DQB1 is depicted in SEQ.ID. NO: 48; and (c) said third conserved primer of HLA-DQB1 is depictedin SEQ. ID. NO: 10 or SEQ. ID. NO:
 12. 7. The method according to claim3 wherein:(a) said conserved oligonucleotide antisense primer ofHLA-DQB1 is depicted in SEQ. ID. NO: 1; (b) said first conserved primerof HLA-DQB1 is depicted in SEQ. ID. NO: 1 and said first nonconservedprimer of HLA-DQB1 is depicted in SEQ. ID. NO: 49; and (c) said thirdconserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 10 or SEQ. ID.NO:
 12. 8. The method according to claim 3 wherein:(a) said conservedoligonucleotide antisense primer of HLA-DPB1 is depicted in SEQ. ID. NO:25; (b) said first and second conserved primers of HLA-DPB1 are depictedin SEQ. ID. NO: 25 and SEQ. ID. NO: 25; and (c) said third conservedprimer of HLA-DPB1 is depicted in SEQ. ID. NO:
 26. 9. The methodaccording to claim 3 wherein:(a) said conserved oligonucleotideantisense primer of HLA-DPA1 is depicted in SEQ. ID. NO: 31; (b) saidfirst and second conserved primers of HLA-DPA1 are depicted in SEQ. ID.NO: 31 and SEQ. ID. NO: 32; and (c) said third conserved primer ofHLA-DPA1 is depicted in SEQ. ID. NO: 33 or SEQ. ID. NO:
 34. 10. Themethod according to claim 3 wherein:(a) said conserved oligonucleotideantisense primer of HLA-DPA1 is depicted in SEQ. ID. NO: 39; (b) saidfirst and second conserved primers of HLA-DPA1 are depicted in SEQ. ID.NO: 39 and SEQ. ID. NO: 38; and (c) said third conserved primer ofHLA-DPA1 is depicted in SEQ. ID. NO: 40 or SEQ. ID. NO:
 41. 11. Themethod according to claim 3 wherein:(a) said conserved oligonucleotideantisense primer of HLA-DPA1 is depicted in SEQ. ID. NO: 33; (b) saidfirst and second conserved primers of HLA-DPA 1 are depicted in SEQ. ID.NO: 33 and SEQ. ID. NO: 32; and (c) said third conserved primer ofHLA-DPA1 is depicted in SEQ. ID. NO:
 34. 12. The method according toclaim 3 wherein:(a) said conserved oligonucleotide antisense primer ofHLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first conserved primerof HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconservedprimer of HLA-DRB is depicted in SEQ. ID. NO: 42; and (c) said thirdconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID.NO:
 13. 13. The method according to claim 3 wherein:(a) said conservedoligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO:2; (b) said first conserved primer of HLA-DRB is depicted in SEQ. ID.NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ.ID. NO: 43; and (c) said third conserved primer of HLA-DRB is depictedin SEQ. ID. NO: 11 or SEQ. ID. NO:
 13. 14. The method according to claim3 wherein:(a) said conserved oligonucleotide antisense primer of HLA-DRBis depicted in SEQ. ID. NO: 2; (b) said first conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primerof HLA-DRB is depicted in SEQ. ID. NO: 44; and (c) said third conservedprimer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO:
 13. 15.The method according to claim 3 wherein:(a) said conservedoligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO:2; (b) said amplifying has four discrete amplifying steps, and whereinin a first discrete amplifying step said first conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 2 and said second conserved primerof HLA-DRB is depicted in SEQ. ID. NO: 6, and in a second discreteamplifying step said first conserved primer of HLA-DRB is depicted inSEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depictedin SEQ. ID. NO. 42, and in a third discrete amplifying step said firstconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and saidsecond nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 43 andin a fourth discrete amplifying step said first conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 2 and said third nonconserved primerof HLA-DRB is depicted in SEQ. ID. NO: 44; and (c) said third conservedprimer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13 orSEQ. ID. NO:
 5. 16. The method according to claim 3 wherein:(a) saidconserved oligonucleotide antisense primer of HLA-DRB is depicted inSEQ. ID. NO: 2; (b) said first conserved primer of HLA-DRB is depictedin SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB isdepicted in SEQ. ID. NO: 45; and (c) said third conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO:
 13. 17. Themethod according to claim 3 wherein:(a) said conserved oligonucleotideantisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) saidfirst conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and saidfirst nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 46; and(c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO:11 or SEQ. ID. NO:
 13. 18. The method according to claim 3 wherein:(a)said conserved oligonucleotide antisense primer of HLA-DRB is depictedin SEQ. ID. NO: 2; (b) said first and second conserved primers ofHLA-DRB are depicted in SEQ. ID. NO: 2 and SEQ. ID. NO: 5; and (c) saidthird conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ.ID. NO:
 13. 19. A method for determining the genotype of one or more HLAClass II polymorphic gene loci of interest selected from the groupconsisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, DQA, --DPB1 and DPA1 of asubject in a sample containing subject genomic DNA using a combinationof conserved and nonconserved oligonucleotide primers, said methodcomprising:(a) amplifying said genomic DNA by at least one amplifyingreaction to generate sufficient first and second double strandedamplifying reaction products for sequencing, using in a separate firstamplifying reaction for each of said gene loci of interest encoded bythe 3'-end of the alpha 1 domain exon, an oligonucleotide primer paircomprising a first conserved primer and either a second conserved primeror a first nonconserved primer for each of said gene loci of interest;(b) sequencing directly at least one strand of said amplifying reactionproduct for each of said gene loci of interest to produce at least onesequencing ladder for each of said gene loci of interest, using in eachsequencing reaction a third conserved oligonucleotide primer for each ofsaid gene loci of interest whereby a nucleotide sequence is obtained foreach sequencing ladder; (c) comparing said nucleotide sequence obtainedfrom each sequencing ladder to known sequences for each gene locus ofinterest; (d) comparing said nucleotide sequence of each sequencingladder obtained with a conserved oligonucleotide primer pair to thenucleotide sequence of each allele of such gene locus amplified with aconserved/nonconserved oligonucleotide primer pair, whereby the genotypeof one or more HLA Class II gene loci is obtained; (e) wherein:i) saidfirst and second conserved primers of HLA-DQB1 are depicted in SEQ. ID.NO: 8 and SEQ. ID. NO: 9, and ii) said third conserved primer ofHLA-DQB1 is depicted in SEQ. ID. NO: 12; (f) wherein:i) said first andsecond conserved primers of HLA-DPB1 are depicted in SEQ. ID. NO: 27 andSEQ. ID. NO: 28, and ii) said third conserved primer of HLA-DPB1 isdepicted in SEQ. ID. NO: 29 or SEQ. ID. NO: 30; (g) wherein:i) saidfirst and second conserved primers of HLA-DPA1 are depicted in SEQ. IDNO: 35 and SEQ. ID. NO: 36, and ii) said third conserved primer ofHLA-DPA1 is depicted in SEQ. ID. NO: 37; and (h) wherein:i) said firstand second conserved primers of HLA-DRB are depicted in SEQ. ID. NO: 20and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID.NO: 5, and ii) said third conserved primer of HLA-DRB is depicted inSEQ. ID. NO: or SEQ. ID. NO:
 16. 20. The method according to claim 19wherein:(a) said first and second conserved primers of HLA-DPB1 aredepicted in SEQ. ID. NO: 27 and SEQ. ID. NO: 29; and (b) said thirdconserved primer of HLA-DPB1 is depicted in SEQ. ID. NO:
 30. 21. Themethod according to claim 19 wherein:(a) said first conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconservedprimer of HLA-DRB is depicted in SEQ. ID. NO: 43; and (b) said thirdconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 13 and SEQ. ID.NO:
 16. 22. The method according to claim 19 wherein:(a) said firstconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and saidfirst nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 44; and(b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO:13 or SEQ. ID. NO:
 16. 23. The method according to claim 19 wherein:(a)said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID.NO: 42; and (b) said third conserved primer of HLA-DRB is depicted inSEQ. ID. NO: 13 or SEQ. ID. NO:
 16. 24. The method according to claim 19wherein:(a) said amplifying has four discrete amplifying steps andwherein in a first discrete amplifying step said first conserved primerof HLA-DRB is depicted in SEQ. ID. NO: 20 and said second conservedprimer of HLA-DRB is depicted in SEQ. ID. NO: 5, and in a seconddiscrete amplifying step said first conserved primer of HLA-DRB isdepicted in SEQ. ID. NO: 20 and said first nonconserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 43, and in a third discreteamplifying step said first conserved primer of HLA-DRB is depicted inSEQ. ID. NO: 44 and in a fourth discrete amplifying step said firstconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and saidthird nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 42; andb) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 13or SEQ. ID. NO:
 16. 25. The method according to claim 19 wherein:(a)said first and second conserved primers of HLA-DRB are depicted in SEQ.ID. NO: 13 and SEQ. ID. NO: 22; and (b) said third conserved primer ofHLA-DRB is depicted in SEQ. ID. NO:
 22. 26. The method according toclaim 19 wherein:(a) said first conserved primer of HLA-DRB is depictedin SEQ. ID. NO: 17 and said first nonconserved primer of HLA-DRB isdepicted in SEQ. ID. NO: 18; and (b) said third conserved primer ofHLA-DRB is depicted in SEQ. ID. NO:
 19. 27. The method according toclaim 19 wherein:(a) said first conserved primer of HLA-DRB is depictedin SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB isdepicted in SEQ. ID. NO: 45; and (b) said third conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 21 or SEQ. ID. NO:
 13. 28. Themethod according to claim 19 wherein:(a) said first conserved primer ofHLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconservedprimer of HLA-DRB is depicted in SEQ. ID. NO: 46; and (b) said thirdconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 21 or SEQ. ID.NO.
 13. 29. A method according to claim 3 wherein:(a) said synthesizingstep and said amplifying step for all of said gene loci of interest areperformed using conserved oligonucleotide primers; and (b) saidsynthesizing step and said amplifying step for all of said gene loci ofinterest are performed in the same reaction tube.
 30. A method accordingto claim 19 wherein:(a) said amplifying step for all of said gene lociof interest is performed using conserved oligonucleotide primers; and(b) said amplifying step for all of said gene loci of interest isperformed in the same reaction tube.
 31. A method according to claim3-30 wherein:(a) each of said synthesizing steps for all of said geneloci of interest are performed simultaneously; (b) each of saidamplifying steps for all of said gene loci of interest are performedsimultaneously; and (c) each of said sequencing steps for all of saidgene loci of interest are performed simultaneously.
 32. A methodaccording to claim 3-30 for rapid automated determination of HLA ClassII genotype of said gene loci of interest of a subject in a samplecontaining subject nucleic acid, wherein:(a) said isolating step isperformed with an RNA/DNA extractor; (b) said amplifying step isperformed by at least one amplifying reaction using a thermocycler; (c)said sequencing step is performed in an automated sequencing apparatuswith a sequencing enzyme and a conserved primer specific for each locusto be sequenced; and (d) said comparing is performed with a computerhaving a data base with allelic sequence information.
 33. A methodaccording to claims 3-30 for rapid automated determination of HLA ClassII genotype of said gene loci of interest of a subject in a samplecontaining subject nucleic acid, wherein:(a) said isolating step isperformed with an RNA/DNA extractor wherein all isolating steps for allgene loci of interest are performed simultaneously; (b) said amplifyingstep is performed by at least one amplifying reaction using athermocycler wherein all amplifying steps for all gene loci of interestare performed simultaneously; and (c) said sequencing step is performedin an automated sequencing apparatus with a sequencing enzyme whereinall sequencing steps for all gene loci of interest are performedsimultaneously; and (d) said comparing is performed with a computerhaving a data base with allelic sequence information.
 34. The method ofclaims 3 or 19 wherein said comparing is conducted with a computerhaving a program including nucleotide sequences of all alleles of allknown haplotypes for all of said loci to be genotyped.
 35. The method ofclaims 3 or 19 where said amplifying with said conserved oligonucleotideprimer includes annealing said conserved oligonucleotide primer of saidisolated nucleic acid at about 37° C.
 36. The method of claim 3 whereinsaid amplifying with said non-conserved primer includes annealing saidnon-conserved primer to said cDNA at about 55° C.